Building a Phenotype Flashcards
(1299 cards)
1
Q
Regulation– Keeping the System in Balance
A
2
Q
Why is regulation required?
A
Different demands on the cell at different times. If there wrre no control, all glucose would be used in respiration, instead of some being used to make other biomolecules, e.g., nucleic acids and fatty acids.
3
Q
2 Methods of Regulation
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- Regulation by gene expression: controlling the amounts of enzyme present. 2. Regulation by fine-tuning the activity of pre-existing enzymes.
4
Q
Controlling the expression of enzymes in a pathway:
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E.g., the lac operon. Slow.
5
Q
Lac Z
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Encodes beta-galactosidase.
6
Q
Lac Y
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Encodes beta-galactoside permease, a transporter that pumps lactose into the cell.
7
Q
Lac A
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Encodes beta-glactoside transacetylase, an enzyme that transfers as ancetyl group from acetyl-CoA to beta-galactosides.
8
Q
Faster response than gene transcription…
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…faster kinetic activity of enzymes, e.g., experimental dtaa demonstrated that E. coli enzyme activity is not wholly gene-dependnet and responds to changes in substrate (glucose) concentration.
9
Q
Regulation by mass action:
A
The rate of enzyme activity being dependent on substrate concentration allows enzymes to respond to changes in substrate concentration, but this is limited.
10
Q
Example of regulation by mass action:
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The enzyme with the lowest Km has the highest affinity for the substrate, so works fastest then reaches a steady state. If [glucose] increases by 10x, the enzymes can work 10x faster. More substrate availabale means it takes longer to reach the steady state. If [glucose] increases by 10x again, it can’t work faster as Vmax has been reached.
11
Q
Regulation by negative feedback:
A
E.g., negative feedback regulation of aspartate transcarbamoylase (ATCase) to produce the right amount of CTP (cytidine triphosphate).
12
Q
Negative Feedback Regulation by Allosteric Inhibition of ATCase
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High CTP concentration inhibits, ATCase. This is allosteric inhibition because CTP binds to a regualtory subunit instead of the active site.
13
Q
ATCase doesn’t display a Michaelis-Menten hyperbolic curve:
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Due to allosteric inhibition. It’s response to substrate concentration is sigmoidal. Pro: very large cahnge in activity within a narrow substrate concentration range.
14
Q
ATCase structure
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6 catalytic subunits arranged as trimers atop one another. 6 regulatory subunits arranged as three dimers that sit in-between the catalytic subunit junctions. CTP binds to a site on each regulatory subunit. The substrate binds at the junctions between catalytic subunits.
15
Q
ATCase Active Sites
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Each catalytic trimer has 3 active sites. Active sites are the junctions between catalytic subunits.
16
Q
Substrate binding and inhibitor binding favour different conformational states.
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Less active T state favoured by CTP binding. More active R state favoured by substrate binding.
17
Q
T state
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Catalytic triads are brought closer together.
18
Q
R state
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Catalytic subunit rotation brings the active site residues closer together for more efficient catalysis. Catalytic triads are 1.2 nm further apart for substrate binding.
19
Q
What solves the energy vs. carbon skeleton partitioning problem in glycolysis in E. coli?
A
Allosteric activation and inhibition of a single enzyme: phosphofructokinase (PFK). Phosphoenolpyruvate binds allosterically to inhibit PFK to favour C skeleton formation. ADP promotes PFK activity, so that more ATP is synthesized.
20
Q
What does Phosphofructokinase catalyse?
A
The conversion of fructose-6-phosphate to fructose-1,6-diphosphate.
21
Q
Regulation by spatial organisation of enzymes:
A
E.g., formation of a purinosome is associated with a 50% increase in the rate of purine biosynthesis from inosine monophospahte (IMP).
22
Q
Organisation of enzymes into a cluster accelerates pathway flux:
A
Local concentration of enzyme is increased. Vmax = [E] x Kcat.
23
Q
Purine Synthesis Pathway
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10 steps, 6 enzymes.
24
Q
What happens if the cell is deprived of purines?
A
Purinosomes form: prine biosyntehsis enzymes arrange themselves in discrete locations, evidenced by fluorescent labelling.
25
Organisation of enzymes into clusters prevents pathways competing for intermediates.
AIR is involved in both the thiamine and purine biosynthesis pathways. Purinosomes prevent AIR from diffusing away into the thiamine biosynthesis pathway because the enzymes ae concentrated locally, so AIR is more likely to move from one enzyme to the next.
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AIR
5-aminoimidazole ribonucleotide.
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Another form of enzyme regulation:
Post-translational modification of enzymes. PTMs extend the properties of amino acids as there are only 20 amino acids.
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Pyruvate Decarboxylase
Its activity is controlled by phosphorylation. Less active when phosphorylated. The kinase is inhibited by pyruvate and ADP.
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Phosphorylation
Typically on amino acids with hydroxyl groups on their side chains: Ser, tyr and Thr. A kinase replaces the H with a phosphate group. The polar group has become doubly negatively-charged.
30
PFK Regulation in Humans vs. E. coli:
Citrate inhibits PFK in humans. PFK is ATP-dependent, so requires ATP to function. High [ATP] in humans inhibits PFK by binding to a different site and promoting C skeleton formation. AMP, instead of ADP, promotes PFK in humans.
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AMP promotes PFK in humans. ADP promotes PFK in E. coli. Why?
Human shave adenylate kinase that converts two ADP molecules to AMP + ATP to top up ATP supply. This means AMP accumulates.
32
What happens without ATP salvage by adenylate kinase?
[ADP] increases by 30% when 10 ATP are used up. With ATP salvage, [AMP] increases by 100% which is greater than the cahnges in [ADP] without ATP salvage, so there's a larger potential impact on PFK amplification.
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What consequences does the amplification of the response due to ATP salvage have?
Humans are more sensitive to ATP depletion than E. coli, which is important to humans due to their graeter metabolic rates.
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35
Introduction to the Organelles of the Eukaryotic Cell
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What % of the cell volume do organelles occupy?
0.5
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Cytoplasm =
Cytosol + liquid.
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Mitochondria
energy metabolisma nd other metabolic functions, incl. synthesisi and degradation of some lipids and synthesis of cofactors such as heme.
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ER
Synthesis of specific proteins, lipid synthesis and protein modification. Some proteins and lipids are sent to the Golgi.
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Vacuole
Salt and other toxic compounds are compartmentalised here to prevent damage to the cell.
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Peroxisomes
Site of oxidative metabolism.
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Endosomes
A series of organelles that endocytosed particles pass through to reach the lysosome/vacuole.
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Lysosomes/vacuoles
contain digestive enzymes to degrade defunct organelles, endocytosed particles and other macromolecules.
45
How much greater in volume is a eukaryotic cell than an E. coli cell?
1,000-10,000x greater.
46
Why did organelles evolve?
Larger volume requires a larger energy supply. Larger volume also means a lower SA:V ratio. Thus, this energy supply couldn't be fulfilled by respiration across the cell membrane alone-- oragnelles with invaginated membranes evolved. Organelles alos enable compartmentalisation of different functions, keeping enzymes in separate places to prevent pathways competing with each other.
47
How did organelles evolve?
Invaginations of the plasma membrane. Connections to the exterior break and the membrane is endocytosed. This forms the nuclear envelope, ER, Golgi, endosomes and lysosomes-- all by similar processes.
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What do the shared origins of organelles mean?
Organelles are topologically equivalent and can fuse to one another, facilitating the transport of cargo without transporter proteins as vesicles bud off the membranes instead.
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Topologically equivalent compartments in…
…the secretory and endocytic pathways.
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Topologically Equivalent
Molecules can travel from one compartment to another without having to cross a membrane or the cytosol.
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The mitochondria-late view assumes evolution of a large genome prior to engulfing the bacterium.
However, a large genome requires lots of energy to maintain.
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Was the engulfing archaeon aerobic or anaerobic?
The syntrophy hypothesis argues anaerobic.
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Mitochondria-late Hypothesis
Loss of the rigid cell wall in the archaeon, facilitating HGT. Phagocytosis and digestion of other prokaryotes increases HGT and so the speed of evolution. Membrane enclose the archaeon chromsome to protect it. An aerobic bacterium is taken up as a symbiont. Multiple mitochondria facilitates membrane systems and larger cells.
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Matrix and stroma
Evolved from bacterial cytosol.
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Endosymbiotic origins of mitochondria and chloroplasts
Isolate them from the vesicle trafficking network, hence their dedicated transport systems.
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Mitochondria and chloroplasts maximise their capacity for energy metabolsim.
Animal cells have 100s of mitochondria; plant cells have 10s of chloroplasts. Chloroplasts have densely-packed thylakoids and mitochondria have extensive membrane invaginations.
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Respirasome
Complete ETC supercomplex.
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Conserved Multiprotein Complexes
MICOS and OPA1 create the junction between the crista and the rest of the intermembrane space.
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ATP synthase dimers
Found at the tip of the crista. The angle between the two ATP synthases determines the curvature of the membrane.
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ETCs are ararnged along the side of the crista.
They interact with each other to form supercomplexes of varying composition.
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Why are crista arranged as they are?
To funnel protons to the ATP synthase dimer, and prevent leakage of protons back across the crista membrane by packing the sides of the crista with ETC supercomplexes for insulation. E- transfer can also occur easily between the tightly packed ETC complexes.
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OPA1 Oligomers
Protein complex that isolates the crista complex like a seal to prevent diffusion. If the crista were not sealed, the malfunctioning of one crista that had lost its conformation would spread.
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PSII dominates the grana.
PSII acts as a pin to stack the thylakoids. PSII is less efficient than PSI as it involves complex chemistry, e.g., photolysisi, so unlike PSI, PSII requires a dedicated structure.
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Where are PSI and ATP synthase found?
On the edge of the membrane, so the NADPH and ATP they produce can diffuse out into the cell.
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Proplastid
Differentiates into various plastid types.
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Etioplasts
Chloroplast progenitors.
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Gerontoplasts
Senescing chloroplasts.
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Desiccoplasts
Occur in desiccation-tolerant plants.
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Chromoplasts
Synthesize and store carotenoid pigments in fruits, flowers and some root tissues.
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Leucoplasts
colourless plastsids defined by the biomolecule they store or synthesize. Incl. Elaioplasts, Proteinoplasts and Amyloplasts.
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Elaioplasts
Store lipids.
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Proteinoplasts
Store proteins.
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Amyloplasts
Important in sesning gravity and determining root growth accordingly. Store starch (amylose/amylopectin) in roots/tubers.
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A chloroplast is a plastid that has differentiated to perform photosynthesis.
If the palstid is in a tissue that will become mesophyll cells, it will receive developmental and light signals directing it to differentiate into a chloroplast.
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Chloroplast genome of Arabidopsis thaliana
117 genes: 87 encode proteins, 8 encode rRNA and 37 encode tRNAs.
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Human Mitochondrial Genome
37 genes: 13 encode respiratory chain subunits, 22 encode tRNAs and 2 encode rRNAs.
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Cyanobacterium Genome
Over 3000 genes!
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Ever since the endosymbiotic event, ...
…there has been transfer of genes fron the chloroplasta nd mitochondrial genomes to the nuclear genome. Chloroplasts and mitochondria cannot carry out their functions without genes from the nucleus.
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TOM
Translocase of the Outer Mitochondrial membrane
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TIM
Translocase of the Inner Mitochondrial membrane
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Additional Problem for Protein Import in Chloroplasts
Proteins must be brought across the thylkaoid membrane as well.
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TOC
Translocase of the Outer Chloroplast Membrane
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TIC
Translocase of the Inner Chloroplast Membrane
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4 Different Pathways for Thylakoid Import
1. Sec pathway. 2. SRP-like pathway. 3. TAT (twin arginine translocation). 4. Spontaneous insertion pathway.
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What is required to import proteins into chloropalst thylakoids?
2 sequential signal peptides: the first signal peptide is excised to reveal the second signal peptide, which is recognised by the receptor in the thylkaoid membrane. Components of prokaryotic origin.
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Sec Pathway
Uses homologues of bacterial Sec proteins, which mediate protein translocation across the bacterial plasma membrane.
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SRP-like Pathway
Uses a chloroplast homologue of the signal recognition particle.
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TAT (Twin Arginine Transloaction) Pathway
Two arginine residues in the signal peptide are critical.
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Spontaneous Insertion Pathway
Seems not require any protein translocator.
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How is the number of mitochondria and chloroplasts in a cell controlled?
By fission and fusion. Chloroplasts divide by fission. Mitochondria divide by fission and can fuse together; they're highly dynamic.
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Why must control the proliferation of chloroplasts and mitochondria?
Prokaryotes normally divide rapidly in reponse to nutrient concentration, which could rupture the cell, if chloroplasts and mitochondria did this.
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Experimental Demonstration of Mitochondrial Fusion
The green fluorescent protein is photoactivated by a laser. Mitochondria labelled with a red fluorscent proteins. When mitochondria fuse, their contents are shared, and they appear yellow.
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How is fusion controlled?
Not directly by the nucleus, but there are some nuclear signals for it.
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How can some mutations in mitochondria be repaired?
By fusion with another mitochondria.
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Outer Membrane Fusion
Caused by an outer-membrane GTPase forms a complex with subunits anchored in the membranes of both mitcohondria.
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Inner Membrane Fusion
A dynamin-related complex forms an oligomeric tethering complex that includes subunits that are anchored in the two inner membranes that are being fused.
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In mammals, where does beta-oxidation of fatty acids occur?
In both peroxisomes and mitochondria.
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Where does beta-oxidation of fatty acids occur in plants and yeast?
Peroxisomes.
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Why are Peroxisomes so-named?
They usually contain oxidative enzymes that use O2 to remove hydrogen from substrates to form H2O2. RH2 + O2 --> R + H2O2
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Why do peroxisomes stand out on electromicrographs?
A crystalloid protein core forms due to the high concentration of oxidative enzymes.
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Peroxisomes contain different enzymes depending on cell type and conditions.
E.g., yeasts grown on sugar have few, small peroxisomes, whereas methylotrophic yeast have numerous, large peroxisomes to oxidise methanol. When yeast are grown on fatty acids, they develop many large peroxisomes to break down fatty acids by beta-oxidation.
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Photorespiration
Occurs in peroxisomes, the detoxification of 2-phosphoglycolate. Exacerbated by high [O2] in the atmosphere. Possible because rubisco can use O2 or CO2 as substrates.
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How are peroxisomes similar to mitochondria and chloroplasts?
Proteins are imported via a translocase pore from the cytosol. New peroxisomes are formed from the fission of peroxisomes using dynamin.
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How are peroxisomes different to mitochondria and chloroplasts?
No genome and only a single membrane, so endosymbiotic origins are unlikley, but may have iriginated by membrane invagination. Some proteins originate in the ER, and ER vesicles can fuse to form precursor vesicles that import the rest of the peroxisomal proteins from the cytosol.
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The evolutionary origins of peroxisomes are unclear.
One theory is that peroxisomes are a vestige of an ancient endomembrane-derived organelle that evolved to use up O2 to keep the [O2] in the rest of the cell low. This may explain the sharing of oxidative metabolism between peroxisomes and mitochondria in modern eukaryotic cells.
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FADH2
Produced in beta-oxidation of fatty acids. Used in mitochondria in oxidative phosphorylation as an electron carrier. Used in peroxisomes to synthesize H2O2 from O2.
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Beta-oxidation of fatty acids
Breaks down fatty acids into acetyl CoA.
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The Endomembrane System I: Key Organelles
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Everything that crosses the plasma membrane
Requires a specific transporter protein.
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Protein Secretion/Inserting Proteins into the Membrane
More complex in eukaryotes than in prokaryotes. Prokaryotes have only a single membrane and NO endomembrane system.
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Signal Peptide
~20 amino acids. Protein sequence at the N-terminus. Very hydrophobic and preceded by positively charged residues. Often removed by a specific protease after secretion.
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The Signal Recognition Particle (SRP): protein secretion in prokaryotes.
The SRP recognises the signal peptide, arresting translation. The SRP promotes association with the protein translocation channel. The SRP dissociates, and the ribosome is attached to the channel. Translation resumes. The nascent protein is translocated co-translationally. Proteases cleave teh signal peptide.
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Transmembrane Domains
Hydrophobic stretches recognised by the translocation channel as start transfer and stop transfer signals. The hydrophobic sections are anchored in the membrane as they are moved out of the translocator into the bilayer.
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Geometric solutions to the SA:V problem in prokaryotic cells:
Cells stay small and rod-shaped.
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SA:V constrains size and shape of prokaryotic cells.
Diffusion-limited processes become inefficient, e.g., if ATP is synthesized on the membrane, it takes longer to diffuse to the centre. Demands of the cytoplasm for ATP and nutrients (r^3) exceed the supply by the plasma membrane (r^2).
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Prokaryotes evolved relatively quickly, …
…whereas eukaryotes evolved more slowly.
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How many eukaryote genes are directly involved in the endomembrane system?
10-20%, i.e., 1,500-6,000 genes.
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Mitochondria and Chloroplasts
Endosymbiotic origin. Double membrane. Function: energy transduction.
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Endomembrane Organelles
Endogenous origin. Single membrane. Function: mport and export of macromolecules.
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Internal membrane-bound organelles
Allow multiple different micro-environments specialised for different functions and increase membrane surface area.
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SRPs are homologous
As they are derived from each other. They contain RNA-- an RNA World relic.
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Protein Translocation in Eukaryotes
The SRP-bound ribosome attaches to the SRP receptor in the ER membrane. Translation continues, and translocation begins. The SRP and SRP receptor are displaced and recycled. The translocation channels are in the ER membrane, so the proteins are translocated into the ER lumen.
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What indicates that the ER evolved from a prokaryotic plasma membrane?
The translocation channel is homologous to the channel in the prokaryotic membrane.
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Rough ER
Continuous with the outer membrane of the nuclear envelope. Studded with ribosomes actively translating and translocating proteins. 1/3 of proteins enter the ER. Controlled environemnt for protein folding.
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Protein folding is a quality control step.
Chaperone proteins in the ER aid folding. Eventually, a signal is added to indicate the protein has been folded correctly. If the protein has not been folded correctly, it will be degraded.
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Many secreted and plasma membrane proteins are modified by glycosylation.
Additions of N-linked glycans (oligosaccharides) to proteins in the ER. The oligosaccharides are linked to nitrogen atoms in Asparagine residues. Glycan processing (i.e., modification of the oligosaccharide) is only completed if the protein is properly folded.
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Functions of Glycosylation
Quality control, signalling and protection.
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Lumen of the ER
Topologically equivalent to the extracellular space because no more membranes need to be crossed to reach the external space.
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Membrane Vesicles
Retain their topology after they bud off. 30-100 nm in diameter. Used for vesicle trafficking.
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Vesicle Trafficking
Budding: lumen proteins are trapped in vesicles and transmembrane proteins are also carried from the donor compartment. Upon fusion to the target compartment, the lumen proteins are released-- either outside the cell or into the lumen of another compartment. The transmembrane proteins are kept the same, so the cytoplasmic side stays cytoplasmic.
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Why don't proteins have to cross any more membranes once in the ER?
Vesicles will transport them between compartments.
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A mixture of proteins can be packaged into one vesicle.
Transmembrane proteins and soluble proteins travel together.
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Protein Export from the ER
Only correctly folded proteins are permitted to leave. They are packaged into transport vesicles that bud off the ER.
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The ER is a continuous membrane system.
The RER is continuous with the SER. All cells contain rough and smooth ER in varying amounts.
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Smooth ER
Site of cellular lipid and sterol synthesis.
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ER
Protein and lipid synthesis. Quality control. Storage. Protein modification.
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Golgi Apparatus
Polysaccharide synthesis. Protein and lipid modification, e.g., lipid glycosylation. Synthesizes glycoplipids and sphingolipids. Sorting.
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Golgi Structure
Stacks of flattened membrane stacks-- cisternae.
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Golgi in Mammals
Stacks form the Golgi ribbon: a large, continuous cluster near the nucleus.
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Golgi in Plants, Fungi and Invertebrates
Many (sometimes 200+) small, mobile Golgi stacks.
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The Golgi processes N-linked glycans on glycoproteins.
By trimming the oligosaccharide to a common core, or by assembly of diverse, complex polysaccharide branches. Indicates where a protein has been, e.g., as an enzyme.
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Camillo Golgi
Was the first to use a stain to visualise the Golgi apparatus in 1898.
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The Golgi stack is functionally compartmentalised.
Cis-, medial- and trans-cisternae harbour different sets of enzymes. Proteins and lipids arrive at the cis-Golgi, pass through the medial and proteins, lipids and polysaccharides leave at the trans-Golgi.
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Polysaccharide Synthesis
Occurs progressively in sequential cisternae.
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Where do Golgi-derived vesicles transport cargo?
The plasma membrane.
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Secretion of some proteins can be regulated.
Proteins are directed into specialised secretory vesicles. Vesicles fuse with the plasma membrane only after a signal is received.
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Constitutive Secreyory Vesicles
Secretion is unregulated.
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Examples of protein secretion regulation:
Insulin secretion from pancreatic beta cells. Hydrolase secretion from pancreatic acinar cells.
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Purpose of the Endocytic Pathway
Transports macromolecules from the external environment to lysosomes/vacuoles for digestion.
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What is transported via the endocytic pathway?
Nutrients (proteins, cells etc.), signalling molecules and pathogens.
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Multiple Internalisation Mechanisms
Phagocytosis by engulfment. Endocytosis via small vesicles.
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Endocytosis
Takes up smaller molecules than phagocytosis. Selective uptake: specific receptor proteins bind to the molecules or cells to be endocytosed-- cargo. Receptors have high affinity for the cargo. The formation of a receptor-cargo complex induces endocytic vesicle formation.
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What compartments do endocytic vesicles fuse with?
Early endosomes.
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Why do early endosomes have a pH slightly lower than the cytosol?
The lower pH affects protein binding, so induces the dissociation of the receptor and the cargo.
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What happens to receptors after they dissociate?
They are packaged into recycling endosomes that fuse with the plasma membrane to return the receptors there.
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The early endosome matures into a late endosome.
Late endosomes also have a slightly lower pH, and are also known as multi-vesicle bodies as they can form internal vesicles within them.
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Where do all the endosomes and enzymes in the endocytic pathway com efrom?
The secretory pathway.
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Lysosomal/vacuolar Enzyme Trafficking
ER --> Golgi --> Late endosome --> Lysosome/vacuole.
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Lysosomes are acidic and hydrolytic.
Low pH degrades substrates. Contains digestive enzymes (proteases, lipases; glycosidases etc.) that are only activated at low pH, otherwise they would degrade the ER and Golgi. The endocytic pathway becomes increasingly acidified.
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Cellular Digestion by Prokaryotes
Secreted enzymes, which leads to diffusion, so it's less efficient. External digestion of macromolecules. Some of the digestion product was transported into the cytoplasm.
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Cellular Digestion by Eukaryotes
Macromolecules, and even cells, are internalised. Internal digestion in hydrolytic compartments, so everything generated from nutrient degradation is available to the cell.
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Waste
Expelled by exocytosis, or kept in compartments, e.g., the vacuole.
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Cell Walls
Prevent phagocytosis, but not endocytosis.
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Cell wall-containing plants and fungi have vacuoles.
Vacuoles can be up to 90% of the cell's volume. Provide turgor pressure for cell growth as the nutrinets and ions in the vacuoles, cause water to move into them. Vacuoles are hdyrolytic or storage compartments.
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LECA
Last Eukaryotic Common Ancestor
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LECA contained the basic set of endomembrane compartments.
ER, endosomes, lysosomes, vesicle transport and phagocytosis. Vesicles later formed other endomembrane compartments. There may have been golgi in LECA, but it's more likely that the functions existed separately.
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The Endomembrane System II: Vesicles, Signals and Sorting.
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What happens to proteins translocated in the ER?
They move in COP-II coated vesicles to the Golgi. Cargo either go further towards the plasma membrane, or are directed towards the endocytic pathway. If in the endocytic pathway, the cargo fuses with the early endosome that matures into a late endosome, and is ultimately digested in the lysosome.
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What is required for compartmental specialisation?
1. Each compartment maintains a distinct set of enzymes and lipids by sorting resident and cargo molecules. This requires signals on proteins to sort them into the correct transport vesicles or retain them in the compartment. 2. acurate targeting of transport vesicles: signals on transport vesicles to target them to the correct compartments.
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Resident Proteins
Remain in the compartment.
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Cargo Proteins
Continue in the pathway.
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Why is the budding of vesicles energetically unfavourable?
The plasma membrane doesn't have much curvature.
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Vesicles form by deforming the membrane.
Protein coats asssemble on the cytoplasmic surface of membranes. Protein coats are made up of protein coat subunits, which have a high affinity for each other, so they build up spontaneously into a larger structure on the surface of the membrane by molecular self-assembly. When a few of teh coat proteins attch to the membrane, more of the coat proteins attcah too automatically, propagating teh deformation and curvature of the membrane.
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Function of Protein Coats
Deform the membrane, and package cargo.
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Coat proteins are useful in packagining the cargo.
They make it more dense and sometimes select the cargo.
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ERES
ER Exit Side, where proteins are packaged into COP-II coated vesicles for transport.
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Resident Proteins in the ER
E.g., chaperones, glycosidases and proteins key to quality control.
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When there are two categories, how many signals are required?
At least one because 1 signal is sufficient to distinguish between two states. If there were 3 states, 2 different signals would be required.
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Initially, it was thought that a signal would be necessary for secretion.
For decades, scientists searched for a conserved sequence between proteins exported from the ER, but none was found.
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Export from the ER can occur without a signal.
Export occurs by default-- bulk flow.
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2 Lines of Evidence for Export as the Default State
1. Introduction of bacterial proteins and artificial peptides with random amino acid sequences into the endomembrane system. 2. Identification of signals that cause proteins to remain in the ER.
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Why were bacterial proteins introduced into the endomembrane system as evidence for export as the default state?
Bacteria don't have endomembrane systems, so wouldn't have a signal specific to the ER.
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How was the bacterial gene introduced into the ER?
Fuse the gene encoding the bacterial protein with a signal peptide to ensure it enters the ER. Add eukaryotic transcription signals (a poly A tail and a promoter) to ensure it is expressed in eukaryotic cells. Insert it into a eukaryotic cell.
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What happened when bacterial proteins were introduced into the ER?
After a short time, the proteins entered the secretory patwhay and were detected in the extracellular space. This implies that no signal is required for export or secretion, but a signal is required for residency.
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The signal is necessary for ER residency.
Deletion of the signal results in secretion of the protein.
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The signal is sufficient for ER residency.
Transplanting the signal into a secreted protein caused its retention.
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ER Residency Signals
ER transmembrane proteins have short sequences containing arginine or lysine exposed to the cytoplasm. ER lumen proteins have a KDEL (lysine, aspartic acid, glutamic acid and leucine) sequence.
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There are 2 possibilities as to how ER residency signals cause the retention of proteins:
1. Physical retention: only the cargo proteins in COP-II coated vesicles can move from the ER to the Golgi, so selection occurs at the ER. 2. Continuous retrieval from the cis-Golgi: everything can leave in COP-II coated vesicles, but Er resident proteins are recycled back.
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Possibility 2 is correct. There are 2 hypotheses as to why:
There are too many proteins, so a selection ssytem would be too complicated. Or it's a more robust vector system to avodi escapees: incorrect proteins could just pass through teh secretory pathway in possibility 1, but in possibility 2, incorrect proteins can be returned to the ER in the 2nd vesicle.
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ER residency signals are in fact…
… ER retrieval signals!
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Why is possibility one not entirely false?
ER resident proteins are not prevented from leaving, but mechanisms exist to slow down their export.
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What is present in the cis-golgi?
Cargo as well as ER resident proteins, so sorting occurs here.
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How are ER resident transmembrane proteins transported back to the ER?
COP-I coat proteins interact with the retreival signals (short, cytoplasmic sequences containing arginine or lysine), which starts the process of molecular self-assembly and forms a vesicle in which all the transmembrane proteins are packaged.
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How are ER resident lumen proteins transported back to the ER?
KDEL-receptor proteins in the cis-Golgi membrane bind to the KDEL retrieval signal on the lumenal side and have arginine or lysine residues on the cytoplasmic side to which COP-I proteins bind. COP-I vesicles form around the lumen proteins.
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What happens after the vesicle to transport ER resident proteins back to the ER has formed?
COP-I coat proteins dissociate, and the vesicle returns to the ER uncoated.
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Coatamer subunits
Formed by the assmebly of COP-I proteins. Flexible, so they stretch when the membrane stretches. Individually, coatamers have some flexibility, but together they are flexible enough to form a sphere.
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COP-I Triad
Contains 7 different proteins with multiple copies. Pre-assembled in the cytoplasm, and binds to the transmembrane proteins to attach the structure to the membrane.
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What does the flexibility of coatamers allow?
The formation of vesicles of different sizes.
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How are COP-I triads connected?
By flexibly attached domains, allowing self-assembly and propagation of membrane bending.
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ER pH
7
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cis-Golgi pH
6.5
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How is cargo binding to and release from the KDEL-receptor regulated?
Lumenal pH. The KDEL-receptor binds at the acidic pH in the cis-Golgi then dissociates at the neutral pH in the ER.
207
How are Er transmembrane proteins returned?
Simply by being released into the membrane, wheras lumenal proteins with the KDEL sequence are still attaached to the receptor.
208
KDEL-receptor Recycling
The KDEL-receptors are returned to the Golgi in COP-II vesicles.
209
COP-II
Anterograde transport.
210
COP-I
Retrograde transport.
211
Resident enzymes of the Golgi apparatus
Made in the ER, and transported to the Golgi, but no further.
212
Most Golgi resident proteins are membrane proteins with…
A large C-terminal catalytic domain, a single transmembrane domain and a short N-terminal cystolic domain.
213
The transmembrane domain and cystolic tail
The localisation signal.
214
Golgi Resident Proteins
Have a different signal. They are mostly enzymes that catalyse sugar polymerisation and modification.
215
Different compartments have different lipid compositions, which modifies the thickness of the membrane.
The more sterols and saturated phospholipids there are, the thicker the membrane will be.
216
Hydrophobic Mismatch
Occurs, if the hydrophobic transmembrane domian doesn't perfectly fit the thickness of the membrane.
217
The plasma membrane, Golgi and ER have different membrane thicknesses.
Plasma membrane (~7 nm) > Golgi > ER (~4 nm).
218
The transmembrane domains of plasma membrane, Golgi and ER proteins differs.
Plasma membrane (>20 amino acids) > Golgi (~17-20 amino acids)> ER (~15-17 amino acids).
219
The transmembrane domains of Golgi transmembrane proteins are necessary and sufficient for Golgi localisation.
If the transmembrane domain is removed from these proteins, the catalytic domain is secreted. If the transmembrane domain is added to a secreted protein, it will be retained in the Golgi.
220
Selective Internalisation
Some plasma membrane proteins (e.g., nutrient receptors) are selectively internalised from the palsma membrane by endocytosis.
221
Cholesterol Endocytosis
Cholesterol is transported as LDL particles, bound by an LDL-receptor at the plasma membrane and endocytosed.
222
Proteins in the Low-Density Lipoprotein
Determine the size of the LDL particle. Important for receptor binding.
223
What happens when LDL particles bind to LDL receptors?
The LDL receptors are activated. The plasma membrane is deformed into pits coated from the cytoplasmic side by coat proteins that are recruited by the activated receptors. A coated pit forms a Clathrin-Coated Vesicle.
224
All coat proteins dissociate after vesicles are formed.
Otherwise, the coat would prevent fusion with the membrane. Coat proteins are only present for forming the vesicles and selecting the pathway.
225
Clathrin-Coated Vesicles have two layers:
The outer layer is Clathrin, and the inner layer is adaptin.
226
Clathrin Complex
Outer layer of the Clathrin-Coated Vesicle. Bends the membrane.
227
Adaptin Complex
Inner layer of the Clathrin-Coated Vesicle. Selects the cargo by bidning to the cytoplasmic tails of the receptors, e.g., LDL-receptors.
228
Different adaptin proteins…
… mean different cargo can be taken up.
229
Adaptin 2 Proteins
Provide specificity at the plasma membrane.
230
Clathrin Coatamer
Called a triskelion, and is made up of 3 smaller proteins and 3 larger proteins.
231
Clathrin-Coated Vesicle Structure
Clathrin triskelions bind adapatins, and can spontaneously self-assemble into small, closed Clathrin lattices. Extremely stable structure that can achieve a high radius of curvature. The bent structure helps deform the membrane.
232
Clathrin-Coated Vesicles are targeted to the early endosome.
The low pH (6.0-6.5) triggers the dissociation of the receptor and the cargo. There is a sorting step: receptors are packaged into recycling endosomes, and returned to the plasma membrane. Cargo (e.g., LDL) travels to the late endosome and lysosome for degradation.
233
Lysosomal hydrolases are made in the ER…
… then transported to the Golgi, usually as inactive pro-enzymes.
234
What happens to the pro-enzymes in the Golgi?
The hydrolases are sorted from the trans-golgi to the late endosome where they join the endocytic cargo delivered from the early endosome. They all travel together to the lysosome.
235
What makes the pro-enzymes inactive?
Protein covers the active site.
236
How are hydrolases activated in the lysosome?
Proteases cleave the peptide bonds to release the inhibitory part of the protein.
237
Lysosomal hydrolases must have a signal…
… to avoid being secreted, and the signal must be different to the signal of the Golgi resident proteins.
238
Lysosomal hydrolases have a signal patch.
A particular surface of the tertiary structure, instead of a specific, well-defined sequence.
239
Neccessity and Sufficiency
Deletion and transplant.
240
Phosphate Tarnsferases
Enzymes in the cis-Golgi that recognise the signal patch, bind to the lysosomal protein and transfer a phosphate onto the 6-mannose of the N-glycan on the protein.
241
Mannose-6 Modification.
Specific to lysosomal proteins.
242
Mannose-6-phosphate receptors in the trans Golgi
Recognise the mannose-6-phosphate residues, bind to the glycan and activate the formation of clathrin-Coated Vesicles into which the lysosomal proteins are packed.
243
Adaptin 1
Recognises the mannose-6-phosphate receptor-protein complex at the trans-Golgi.
244
pH in the late endosome
More acidic than in the trans-Golgi, facilitating mannose6-phosphate receptor-cargo dissociation. The receptors are recycled back to the trans-Golgi in vesicles.
245
Summary of Vesicle Trafficking
ER to Golgi-- default. Golgi to plasma membrane-- default. Return to ER-- selective, COP-I. Diversion to a lysosome-- selective, Clathrin-Coated Vesicle. Endocytosis-- selective, Clathrin-Coated Vesicle.
246
There are 4 possible fates for proteins that arrive in the Golgi:
Default: transport to the plasma membrane and secretion. The other 3 fates are signalled. Transport to the lysosomal system in a Clathrin-Coated Vesicle. Return to the ER in a COP-I vesicle. Retention in the Golgi.
247
SNARE Proteins
Proteins with a long, coiled domain on the cytoplasmic side.
248
v-SNARE Proteins
Vesicles
249
t-SNARE Proteins
Compartments
250
A different combination of SNAREs…
… exists for each vesicle targeting event.
251
What happens when a vesicle docks?
Proteins test the interactions between the v- and t-SNAREs.
252
trans-SNARE Complex
A coil-coil complex that forms, if the v-SNARE contains information indicating the vesicle should merge with the compartment. This pulls the vesicle close to the membrane, so membrane fusion occurs.
253
NSF Complex
Structurally related to ATPases. Separates upon ATP investment after vesicle-compartment membrane fusion, which separates the v-SNAREs from the t-SNAREs. The v-SNAREs are returned in vesicles.
254
Why must there be a recycling step after membrane fusion?
Mixed identity: there would be a combination of v- and t-SNAREs on the membrane surface.
255
NSF Complex
N-ethylmaleimide sensitive fusion protein.
256
257
Cytoskeleton I: Microtubules
258
259
Internal membranes solve the problem of inadequate membrane surfaces for metabolic reactions, but…
… don't solve the problem of inadequate diffusion for some nutrients and metabolic reactions.
260
Endocytic and Secretory Vesicles
Diffuse very inefficiently from the periphery to the interior.
261
Cytoskeleton
3D transport network that fills the cytoplasm.
262
3 Functions of the Cytoskeleton
Controls cell shape, provides structural support for the cell and drives cell movement-- intracellular organelle and vesicle movement, cell motility and muscle contraction.
263
The cytoskeleton is also responsible for positioning.
E.g., positioning mitochondria closer to the nutreints.
264
3 Classes of Cytoskeletal Elements, all cross-linked together.
All involve long, unbranched; one-dimensional protein polymers. Microfilaments, intermediate filaments and microtubules.
265
Microfilaments
Actin. 7nm. Present in all eukaryotes.
266
Intermediate Filaments
10 nm. Only present in animals, not all eukaryotes, but this is still debated. Cytokeratin, Vimentin and Desmin. An umbrella term for structurally-related protein filaments.
267
Microtubules
Tubulin. 25 nm. Present in all eukaryotes.
268
Filament Propteries
Determine how dynamically they can change.
269
Actin filaments and microtubules are polar
Each end has different properties, so different processes occur at each end, giving directionality to the filament.
270
Intermediate filaments are much less dynamic than microfilaments and microtubules.
They cannot bind nucleotides, and are apolar, so there's no distinction between the two ends.
271
Actin filaments and microtubules can bind ATP and GTP respectively.
There is energy investment in the process meaning both filaments are highly dynamic, changing structures.
272
All 3 filaments can spontaneously self-assembly.
This self-assembly occurs in vivo and in vitro.
273
How are intermediate filaments apolar, if they are made up of proteins with coil-coil domains with distinct ends?
The proteins homo or heterodimerise, but still retain their distinct ends. When the coil-coil domains laterally interact with each other, the dimers are arranged in an anti-parallel position. This forms a tetramer with indistinct ends. The tetramers further laterally associate with other tetramers to form a unit length filament. Unit length filaments with identical ends are assembled into an intermediate filament.
274
Subunit Exchange
Occurs along the length of the intermediate filament instead of at the ends.
275
Functions of Intermediate Filaments
Structural support. Determination and maintenance of cell and nucleus shape.
276
No organelle transport along intermediate filaments.
As they don't have motor proteins.
277
Properties of Intermediate Filaments
Extensible (3.5x its length), high tensile strength; resistant to compression, twisting and bending.
278
Nuclear Lamina
Lattice of nuclear lamins at the interface between the nuclear envelope and chromatin to provide a surface for chromatin anchorage.
279
What consequences does a lack of lamin A have?
Slows cell division, blocks DNA replication, blocks RNA Polymerase II progression and reduces heterochromatin content.
280
What does a point mutation in human lamin A cause?
Premature ageing in children-- Hutchinson Gilford Progeria Syndrome.
281
Tubulin
Alpha-beta heterodimer.
282
Polar filaments can polymerise and depolymerise rapidly.
Extension and shrinkage occurs at the ends as polar filaments are one-dimensional.
283
Microtubule Structure
Tubulin heterodimers associate with each other in a head-tail position. An alpha tubulin binds to a beta tubulin to form a protofilament. 13 protofilaments form a long, hollow cylinder.
284
Beta Tubulin
GTPase that can slowly hydrolse GTP to GDP. Alpha tubulin cannot hydrolyse GTP, but there are binding sites for GTP on both.
285
Tubulin Heterodimer
Microtubule subunit. Subunits are dynamically assembled through GTP hydrolysis into microtubules.
286
Minus End
Alpha tubulin, less dynamic.
287
Plus End
Beta tubulin is exposed, so new subunits are added here. More dynamic.
288
GTP hydrolyssi starts when the subunits are incorporated into the filament at the plus end, but…
… GTP hydrolysis is very slow, so many more subunits have been added by the time GTP is hydrolysed. This means GTP hydrolysis occurs somewhere in the filament and not at the exposed end.
289
GTP hydrolysis causes a slight conformational change in the subunit.
GTP-bound subunits are straight, so the protofilaments can align with each other. GDP-bound subunits are slightly bent.
290
GDP-bound subunits are slightly bent. What does this mean?
If GDP-bound subunits are at the exposed end, the protofilaments bend away from each other, destabilising the stricture. As long as GTP-bound subunits are at the + end, the structure won't disassemble.
291
GTP Cap
Lots of GTP-bound subunits at the + end. This stabilises the filament, so polymerisation can occur.
292
If the incorporation of GTP-bound subunits exceeds the rate of GTP hydrolysis, …
… polymerisation occurs.
293
If the rate of GTP hydrolysis in the filament exceeds the rate of incorporation of new subunits, …
… shrinkage of the filament/depolymerisation occurs.
294
Dynamic Instability
Alternating growth and catastrophe cycles at the plus end due to the kinetics of GTP hydrolysis. It's dynamically moving back and forth.
295
Catastrophe/depolymerisation
Very fast.
296
Why doesn't catastrophe always lead to the complete depolymerisation of the filament?
There may be pockets of GTP-bound subunits that slow depolymerisation and provide time and a surface for new GTP-bound subunits to be incorporated, allowing the filament to grow.
297
Where do microtubules continually grow from?
The centrosome.
298
Nucleation
Birth of the filament. Unlikely to occur spontaneously as it is energetically unfavourable to organise 13 protofilaments into a cylindrical structure.
299
Why is the minus end more static?
It is frequently blocked by the large gamma-tubulin ring complex.
300
Gamma-tubulin Ring Complexes
Initiate filament nucleation by providing a building surface.
301
Gamma Tubulins
Structurally related to alpha and beta tubulins. Held in place by other proteins. Gamma tubulins preferentially bind to alpha tubulins, so bind at the - end.
302
13 Gamma Tubulins
Constitute the intiation platform for the filament.
303
Gamma-tubulin ring complexes are anchored to organelles.
This forms a network of filaments with a determined polarity.
304
Small Complexes
The smaller gamma-tubulin complexes found in plants.
305
Where do the small complexes bind/associate in plants?
Small complexes asssociate with the nuclear envelope, so the microtubule network irradiates from the nucleus. In mature cells, the small complexes associate with the cell edges, so the microtubules are arranged in a cortical manner just under the membrane.
306
Microtubules guide the direction of cellulose polymerisation in the cell wall.
Enzyme complexes in the plasma membrane extrude cellulose as they travel along the cortical microtubules.
307
How many microtubules in interphase in animal cells?
~50
308
Microtubule Organising Centre
The centrosome, which contains 2 centrioles. Gamma-tubulin ring complexes asssociate with the surface of the centrioles.
309
How are microtubules organised in interphase in animal cells?
Centrosomes are localised next to the nucleus, so the microtubule array penetrates all of the cytoplasm with the + ends at the edges of the cell.
310
Some microtubules are static. Others undergo dynamic instability. Why is this important?
Dynamic instability allows the cell to explore or sense changes in the cytoplasm or at the plasma membrane.
311
What happens when the microtubule reaches the plasma membrane?
It is at an unfavourable angle, so loses the GTP cap and undergoes depolymerisation/catastrophe. Then it is rescued and grows again. It grows back and forth, probing the plasma membrane.
312
When is it important to be able to sense local changes at the plasma membrane?
When the cell changes shape or during cell movement.
313
Most microtubules are regulated by MAPs.
Microtubule Associated Proteins: stabilises the filament by binding to the GTP cap to keep it intact, even when GTP hydrolysis occurs, so the filament doesn't undergo catastrophe and continuous growth is possible.
314
Catastrophin Proteins
Bind to the GTP cap to increase the bend in the protofilament, leading to catastrophe.
315
In animal cells, what are microtubules responsible for during interphase?
Moving organelles and moving COP-II vesicles from the ER to the Golgi, moving vesicles through the Gogli and to the plasma membrane and movement through the endosomal system.
316
What are microtubules responsible for during mitosis in animal cells?
Chromosome movement.
317
Motor proteins achieve bidirectional transport.
Dyneins and kinesins.
318
Dyneins
Walk towards the minus end. Carry COP-II proteins from the ER to the Golgi.
319
Kinesins
Walk towards the plus end, usually. Return COP-I vesicles to the ER.
320
Location of the ER
At the periphery.
321
Location of the Gogli
At the centre, next to the Microtubule Organising Centre.
322
Structure of Kinesins
Homodimers with a stak region containinga slightly broken coil-coil domain, a cargo-binding tail domain and two motor heads at the opposite end to the cargo-binding domain.
323
8 nm
Length of a tubulin subunit.
324
How do kinesins walk along the microtubule?
The kinesin binds to the microtubule and takes an 8 nm-long step. Each step requires the hydrolysis of ATP to provide energy for movement. It occurs in the same way each time. Kinesins move processively, step-by-step, along the mcirotubule.
325
What do each of the two identical kinesin motor heads consist of?
A catalytic core and a neck linker. Each haed also contains ADP.
326
Kinesins move randomly by Brownian motion, but what happens when one of the motor heads encounters a microtubule?
It binds tightly, releasing the ADP. ATP can then bind instead.
327
What does the nucelotide exchange trigger?
The neck linker to zip onto the catalytic core. This throws the 2nd head forward to the next binding site on the microtubule.
328
What happens after the 2nd head binds to the microtubule?
The 2nd, trailing head becomes the leading head. The trailing head hydrolyses ATP to release a phosphate. The neck linker detaches from the trailing head. Ncuelotide exchange (ADP for ATP) at the leading head. The cycle repeats.
329
What does ATP binding cause?
A conformational change that means the motor head advances by one step then throws the trailing head forward.
330
What does ATP hydrolysis cause?
Release from the microtubule.
331
Dynein Structure
Built from many different proteins, but one end still binds to the cargo and the other end binds to the microtubule.
332
Dynein movement is erratic.
Sometimes on step forward, sometimes one step back; sometimes sideways. Step length is inconsistent, so it's not as well understood as kinesins.
333
Accessory Proteins
Allow dynein proteins to bind to transport vesicles.
334
What powers dynein movement?
ATP hydrolysis at the dynein motor head.
335
Microtubules can push and pull organelles.
Microtubules are 150-fold more rigid than microfilaments, so transmit compressive as well as tensile forces.
336
Spindle
A microtubule filament irradiating from a spindle pole body.
337
Function of Spindles
To position and push spindle poles. To pull sister chromatids to opposite poles and contract chromosomes.
338
How does spindle separate the two daughter cells?
Spindles link the two spindle poles, and push against each other.
339
How does spindle pull sister chromatids to opposite poles?
Chromosomes attach at the + end, then are pulled in as microtubules shorten.
340
Eukaryotic cilia and flagella
Structurally identical cellular extensions containing complex arrays of microtubules, hence 'cilia' is often used to refer to both to avoid confusion with bacterial flagella.
341
Ciliated/flagellated plants
Hava basal bodies, but not centrioles.
342
Basal Bodies
arrays of microtubules. Originate from centrioles in interphase cells. Cilia and flagella grow from basal bodies.
343
Eukaryotic cilia and flagella are intracelullar structures.
The plasma membrane coveres the whole structure. The axoneme is the microtubule.
344
9 + 2 Arrangement
The 9 are outer doublets. Each doublet is comprised of an A microtubule and a B microtubule. The 2 are central singlets.
345
Microtubule A
13 Protofilaments.
346
Microtubule B
11 Protofilaments.
347
Nexins
Interconnected proteins that link doublets together.
348
Spike Proteins
Attach the doublets to the centre.
349
Ciliary Dynein
Large protein complex with 3 motor heads instaed of 2. Attached to the surface of the A microtubules, but walk along the surface of B microtubules when ATP is hydrolysed, causing bending.
350
Not all the dyneins can be active at the same time.
Waves of activation of the dyneins leads to waves of bending. Propagation of the bending activity down the flagellum leads to a sinusoidal waveform.
351
What would happen, if the microtubules were not attached by the cross-linking nexin proteins?
The microtubules would slide instead of bend when the dyneins walk.
352
Motility in Cilia
Short. The asymmetric beat creates a force perpendicular to the longitudinal axis, which is useful when moving substrate in the small intestine.
353
Motility in Flagella
Long. The usually symmetric sinusoidal beat generates a force parallel to the longitudinal axis, so it moves forward.
354
Eukaryotic Flagella
Made of tubulin. Intracellular. Moves by coordinated dynein movement.
355
Bacterial Flagella
Made of flagellin. Extracellular. Moves via a rotatory motor complex.
356
Did LECA have flagella?
LECA likely had two cilia/flagella with sensory functions.
357
Vertebrate Retina Cells
Have short, cilia structures that connect the outer and inner segments. Key to signal transduction.
358
Olfactory Neurones
Have lost their motility, but still have several cilia to detect smells and signals.
359
Sperm
Sense the environment, and move in a particular direction accordingly.
360
Cilia evolved in unicellular organisms, so…
… they had to sense the environment to determine the direction of movement as movement was governed by extracellular signals.
361
Receptors, ion channels and transporter proteins
Localise to the cilium.
362
363
Cytoskeleton II: Actin-Based Cell Movements
364
365
G-actin
Globular actin. Microfilament monomer.
366
F-actin
Filamentous actin. Microfilaments are helical polymers.
367
Actin filaments work like ropes:
Better for pulling than pushing as they are strong in tension, but weak in compression.
368
Microfilament Structure
A left-handed helix with a rotation of 166° per subunit. 13 monomers comprise one helix repeat of 37 nm. Diameter: 7 nm.
369
G-actin binds 1 ATP in the nucleotide-binding cleft.
The hydrolysis of ATP means there is energy investment in this process.
370
G-actin has a different surface exposed at each side.
Subdomain 2-4 is close to the nucleotide binding cleft; subdomain 1-3 surface is on the other side.
371
How does polarity arise in the microfilament?
The 1-3 surface always binds to the 2-4 surface when G-actin polymerises.
372
At which end does polymerisation occur?
The + end as the + end has the highest binding affinity to ATP-binding G-actin.
373
What happens when G-actin is incorporated into F-actin?
After a delay, ATP is hydrolysed, and the phosphate is released, which destabilises the filament.
374
How does ATP hydrolysis destabilise the filament?
It results in a slight conformational change, so ADP-bound subunits are less well cross-linked and have weaker interactions between them than the ATP-bound section. The interactions are still strong enough to keep it from falling apart.
375
What happens, if a ADP-bound subunit is exposed at the - end?
The preferential dissociation of ADP-bound subunits at the - end occurs.
376
Treadmilling
The actin filament elongates at the + end, and depolymerises at the - end.
377
What allows G-actin to be built into F-actin?
G-actin has a higher binding affinity for ATP than ADP, so ADP is automatically exchanged for ATP, if there's sufficient ATP in the cytoplasm.
378
Nucleators
Proteins that start the nucleation of the filament.
379
Capping Proteins
Stabilise the filament, but don't allow further growth as they atatch to the + end. Important in muscle movement.
380
Severing Proteins
Bind to older, ADP-bound stretches and cleave/sever them, so depolymerisation of the newly-exposed ends occurs.
381
Sequestering Proteins
Sequester monomers by binding to them, so they cannot be incorporated into the filament.
382
Bundling Proteins
Build up larger structures.
383
Actin binding Porteins
Determine the rate of filament assembly and stability.
384
Functions of actin binding proteins:
Nucleation, capping, severing, sequestering and bundling.
385
Function of microfilaments in plants and fungi:
Linear pathways for organelle movement.
386
Function of microfilaments in animals:
They form contractile systems together with motor proteins to ensure contraction in the cell during cell movement and cell division. When cross-linked, they can have a variety of structural roles, and can push the growing margins of the animal cell forwards.
387
Why are mysosins structurally similar to kinesisn?
They have the same evolutionary origin at the time of early eukaryote evolution.
388
Myosins
A family of motor proteins that allow movement and contraction along the actin.
389
Myosin Structure
2 motor heads, each binding ATP, that are connected by a neck linker to the tail, which is a coiled-coil domain. At the C-terminus of the tail, the myosin can bind to a vesicle or organelle, or the myosins can laterally interact with each other by dimerising.
390
Motor Heads
Convert chemical energy from the hydrolysis of ATP into mechanical movement.
391
Plant cells have the fastest-moving…
… organelles in all kingdoms.
392
Organelle and vesicle transport in plants
Small organelles and vesicles are continuously moved around the cytoplasm on the actin using myosin proteins.
393
Cytoplasmic Streaming
The drag caused by moving organelles causes the whole cytoplasm to cycle round the cell. Required to overcome the diffusion barriers as palnt cells are very large due to the vacuole.
394
Myoblasts
Mononucleated precursors that fuse to form myotubules that are surrounded by a plasma membrane to form muscle fibres.
395
Muscle Fibres
Giant, animal, multinucleate; syncitial cell. ~50 micrometres in diameter. Within each fibre, there are many myofibrils.
396
Contractile Arrays
Formed from actin and myosin-II in animals.
397
Each myofibril
A linear array of sarcomeres.
398
Sarcomere
Contractile unit between 2 Z discs. Small contractions of each sarcomere contracts the muscle overall as sarcomeres are in a linear array.
399
Z discs
Anchorage points to which the + ends of the actin filaments bind. Microfilaments with opposite polarities project from the two Z discs.
400
The thick filament
More strongly stained. A bipolar assembly of myosin-II proteins, which laterally associate via their coiled-coil tail domains. Each end has myosin heads facing in opposite directions and protruding out of the filament.
401
Why are there no myosin heads in the middle of the thick filament at the M line)?
It's just tail domains.
402
Nine Radial Positions
Nine myosin heads protrude out from the filament.
403
Titin and Nebulin
Giant proteins that acts as molecular rulers by controlling the lengths of the thick and thin filaments.
404
Titin
A titin molecule with elastic ends attaches the thcik filaments to the Z discs on each side, so when a force acts on the sarcomere, an equal force is exerted on both sides of the sarcomere, so the thick filaments stay in the centre of the sarcomere.
405
The - ends of the microfilaments in a sarcomere
Have a capping protein, so their length is fixed.
406
What determines the length of the microfilament?
The length of the nebulin protein that wraps around the microfilament, so all the micorfilamnets in the sarcomere are the same length.
407
The sliding filament model of contraction:
Actin filaments slide towards the centre of the sarcomere, so the sarcomere is shortened. The actin is under tension because the myosin molecules bind to the actin and pull it towards the centre of the sarcomere.
408
The identical motor heads in the myosin dimer
Act independently. The coiled-coil rod connects the heads and tethers tehm to the thick filament.
409
Myosin heads before contraction
Have ADP + Pi bound, and a weak affinity for actin.
410
How far does each myosin head move?
6nm
411
Myosin vs. kinesin
With myosin, the heads are not coordinated.
412
Why does the actin not slide back when released from the myosin?
There are many other myosins holding it in place, keeping it under tension.
413
For what % of the hydrolysis cycle is the myosin attached?
0.05
414
The thick filaments are hexagonally packed
6 actin filaments surround 1 myosin filament.
415
The symmetry of the myosin heads and their surrounding myosin actin binding sites differ.
The myosin heads protrude at 9 radial positions, but each thick filament is surrounded by 6 thin filaments.
416
The spacing of the myosin heads does not align with the spacing of the myosin binding sites on the thin filament.
Myosin head repeat distance: 129 nm. Each head moves ~ 6nm per cycle. Repeat distance of the myosin binding sites on the thin filaments: 37 nm.
417
The lack of alignment and symmetry means the myosin heads cannot all bind at the same time.
Ensuring some heads are bound at all times to maintain tension.
418
Thin filament accessory proteins
Troponin and tropomyosin.
419
In the relaxed state:
Tropomyosin is in the groove between the actin filaments, and blocks the myosin binding sites.
420
Troponin Complex
Made up of I, C and T proteins.
421
When 2 Ca^2+ ions bind to troponin C, …
... The troponin complex changes shape, and induces the tropomyosin to roll away from the myosin binding site on the thin filament. Myosin can bind, causing contraction until [Ca^2+] drops.
422
Where is the sarcoplasmic reticulum located?
Between the myofibril and the plasma membrane.
423
How are Ca^2+ ions released?
Action potentials transmitted to the plasma membrane can be transmitted to the sarcoplasmic reticulum as they are in close proximity. This opens Ca^2+ ion channels, increasing [Ca^2+] tenfold.
424
Resting [Ca^2+] in the sarcoplasm
~10^7 molar.
425
[Ca^2+] within the sarcoplasmic reticulum
~10^3 molar.
426
Cytokinesis in plants and fungi
A contractile ring of actin and myosin separates the daughter cells by forming a cleavge furrow. The actin tightens to narrow the ring,a nd pinch apart the two cells.
427
Stress Fibres
Cytoplasmic bundles of F-actin and myosin-II. Contractile actin arrays.
428
Stress fibres are attached at two points to maintain tension.
At one point, they are attached to the plasma membrane at the focal adhesion point. The other point can be another focal adhesion point, or it can be next to the nucleus. Upon a signal, myosins pull the actin on either side, contracting the cell.
429
Focal Adhesion Points
Anchor cells to other cells or to the extracellular matrix. Cells can push away from the focal adhesion point.
430
Stress Fibre Structure
Determines cell shape and cell localisation. Contain mini-sarcomere with bipolar actin filaments and myosin II mini-filaments.
431
Actin polymerisation can drive rapid cell migration.
The actin network directs the movement of keratocytes (wound-healing cells in fish) and crawling cells.
432
3 Different Actin Filament Arrays in Crawling Cells
Lamellipodium, stress fibres and the filopodium.
433
Lamellipodium
Large, flattened cell extension at the leading edge. Pulls the cell forward. Quasi-2D, branched actin meshwork.
434
Filopodium
Temporary structure that probes the environment before the lamellipodium extends there. Small, dynamic cell projections. Actin bundle. Senses environmental signals.
435
Stress fibres in crawling cells.
Attached to focal adhesion points at the trailing edge. When they contract, they push the trailing edge.
436
The lamellipodium of a fish scale keratocyte cell
Contains a dense array of short actin filaments. The short filaments in these dense arrays provide enough rigidity to push on the membrane at the leading edge. 70° angles between short filaments forming a branched network.
437
Branched arrays are organised by Actin Related Proteins (ARPs).
The actin filament itself doesn't branch, but a nucleation point is formed on the side of the filament when the ARP2/3 complex binds at a 70° angle. Creates a cross-linked meshwork. The filament is terminated by capping.
438
ARP2/3 Complex
Similar to G-actin structurally, but cannot polymerise. Instead, it initiates polymerisation. Analagous to gamma-tubulin.
439
Individual actin filaments in a branched, lamellipodium array:
Become capped at both ends.
440
Array Treadmilling Model for Lamellipodium Extension
The array is treadmilling, although the individual filaments are not. The array as a whole grows forward: polymerising at the front due to exposed + ends just beneath the plasma membrane and depolymerising at the rear as severing enzymes cut the ADP-bound actin to expose it. Net filament assembly at the leading edge. Net filament disassembly at the trailing edge.
441
Protrusion at the leading edge is mediated by actin polymerisation.
Protrusion doesn't require motor proteins, e.g., myosin II; it's based of the polymerisation of actin fialments. When the lamellipodium protrudes forward, stress fibres contract, and add a new focal adhesion point further forward.
442
Different GTP-binding G-proteins
Induce the formation of different actin structures in different parts of the cell. Rho, Rac and Cdc-42.
443
3 different types of actin network in the same cell.
Spatially organised and activated in different parts of the cell.
444
Rho
Stimulates stress fibre production. Found at the trailing edge.
445
Rac
Stimulates extension of the lamellipodium.
446
Cdc-42
Stimulates formation of filopodia. Found at the leading edge.
447
G-proteins are molecular switches.
GTPases. In the GTP-bound form, they are active and can interact with many different proteins in actin network formation. After GTP is hydrolysed, they are inactivated, so no longer interact with the relevant proteins. They can be activated agin by exchanging GDP for GTP.
448
GTP binding and release in G-proteins
Controlled by other factors, so their activity can be regulated by environemntal signals.
449
Coordination among cytoskeletal elements
Can occur at regulatory steps, or by cross-linking proteins in different cytoskeletal elements.
450
Static Crosslinkers
The two proteins are continuously linked together.
451
Linking by complexes of motor proteins
One actin and one myosin, so they can each walk along the other filament.
452
Motor protein-binding protein complex
One side is a motor protein and the other side is a fixed attachemnt, so one filament slides along the other.
453
454
Extracellular Matrices
455
456
Extracellular Matrix
3D network of macromolecules surrounding cells that is outside the cell membrane.
457
All cells assemble an extracellular matrix of macromolecules.
Archaeal cell walls, bacterial peptidoglycan cell walls, fungal cell walls of chitin, bacterial outer membrane, plant cell walls and the animal extracellular matrix.
458
Functions of extracellular matrices
Structural support, protection, signalling and regulation of cellular activities.
459
Structural Support
Maintaining cell integrity, tissue organisation and cell adhesion.
460
Protection
First frontier of protection against mechanical, chemical and biotic stresses. They can either reduce the impact of or repel these stresses.
461
Signalling
Perception and transmission of chemical and mechanical signals from the environment. Cell-cell communication.
462
Regulator of cellular activities
Regulates cell growth, motility and morphogenesis.
463
Eukaryotic extracellular matrices are fibre-reinforced composite structures made up of…
… fibrils, fibril cross-linkers and gel.
464
Fibrils
Protect against tensiosn and compression. Insoluble. Provides strength, stiffness and elasticity. Made up of elastin and collagen proteins. Collagen is arranged into porous sheets (basal lamina) and linear fibrils.
465
Fibril Crosslinkers
Organise and strengthen the fibril network. Made up of collagen protein.
466
Gel
The fibril network is embedded in it. A hydrophilic environment. It ensures hydration. Protects against compression. More polysaccharide than protein. Made up of proteoglycans. A core of proteins with attached glycosaminoglycans (GAGs).
467
All cells have extracellular matrices, but not all cells synthesize it themselves.
Fibroblasts synthesize it in connective tissues. Extracellular matrices is a main feature in cells that have a structural function.
468
What is the most abundant protein in the aminal kingdom?
Collagen. 30% of human body weight is collagen.
469
Collagen
Insoluble, fibrous protein. Long, straight and rod-like. Three amino acid repeats where every 3rd amino acid is glycine. The other 2 are either lysine, proline of hydroxyproline, lending stability to the helix.
470
Basic structural unit of collagen
A triple helix. Each chain has ~1050 aminoa cids wound into a right-hand triple helix. Glycines form the hydrophobic inner side. The other 2 amino acids in the triplet are exposed as they are more hydrophilic.
471
Why is hydroxylation important in collagen synthesis?
Key to protein-protein interactions. Interactions between the hydroxylated side chains allow the helix structures to assemble into a long polymer.
472
Why does scurvy result in a lack of connective tissue?
Vitamin C catalyses the hydroxylation step.
473
Collagen helices are cross-linked…
…at specific, regualr positions where they can interact laterally with each other, giving fibrils a striated pattern.
474
Gaps between collagen molecules
The same length and at regular intervals. Stained strongly as the dye gets stuck in the gaps.
475
Fibril-Associated Collagen
Responsible for cross-linking collagen fibrils. In fibril-associated collagen, the triple helix structure is interrupted by non-helical regions.
476
Why are the non-helical regions important in fibril-associated collagen?
It means the polymer structure appears as two rods with a hinge instead of a single rod.
477
Connective Tissues
Cartilage, liagments and tendons. This enables the association of one side of the collagen fibre with another. This determines the network structure. Especially important in connective tissues.
478
Network-forming Collagen
Assembled into a triple helix with non-helical breaks to introduce flexibility. Interact laterally with each other to form networks instead of a long fibre.
479
Type IV Collagen
A type of network-forming collagen. Self-assembles into a sheet-like meshwork to help form basal laminae. The N and C terminal globular domains help stack sheets on top of each other to form a dense mesh.
480
Basal Laminae
Underlies all epithelial cell sheets and tubes, e.g., the kidney glomerulus, and surrounds individual muscle, fat and Schwann cells. Contains additional proteins, e.g., laminins, to help tighten the cross-linking.
481
The basal lamina has structural and filtering roles, and determines cell polarity.
When a tissue attaches to the basal lamina, polarity is provided to the cell.
482
What do basal laminae influence?
Cell metabolism, organisation of proteins in the plasma membrane, induction of cell differentiation and it serves as highways for cell migration.
483
Cell Migration
Occurs frequently on basal laminae. They provide surfaces on top of which to move, but don't allow cells to pass through them.
484
Tropoelastin
Soluble precursor to the elastin fibre. Highly extensible proteins that behave as molecular nanosprings.
485
Tropoelastin molecules are synthesised in the Er, then secreted into the extracellular matrix. What happens to them there?
They are cross-linked at random places into elastin polymers by self-assembly.
486
Elastin
Hydrophobic. Huge potential to expand then regain its original size. Highly disorganised network.
487
Function of Elastin
Provides the elasticity of loose connective tissue, skin, lungs and blood vessels.
488
What is the simplesta nd most abundant GAG?
Hyaluronan/hyaluronic acid made up of 25,000 repeats of glucuronic acid and N-acetylglucosamine.
489
Glycosaminoglycans
Regular repeats of disaccharides: a uronic acid (glucuronic acid or galacturonic acid) and an amino sugar (N-acetylglucosamine or N-acetylgalactosamine).
490
Properties of GAGs
Usually heavily sulphated, giving a negative charge, so they are highly hydrophilic and attracted to positive ions. These polysaccharides take up a lot of water to form a disorganised gel, even at low concentrations. Adopt extended conformations, and occupy a huge volume relative to their mass-- inflexible.
491
GAGs have a high density of negative charge that attracts Na+ ions.
This osmotic effect increases turgor pressure by swelling, enabling it to withstand large, compressive forces.
492
Proteoglycans
GAGs attached to a protein backbone via serine or threonine. These amino acids are glycosylated at the O of the OH group to form a linker to which the polysaccharide is attached.
493
Glycoproteins
Not part of the gel. 1-40% carbohydrate by weight.
494
What % of proteoglycans are carbohydrate?
Up to 95%.
495
Plant cell wall
Synthesised by all plant cells. Fibre-reinforced composite. Analagous to the animal extracellular matrix, but has different physical and chemical properties.
496
Plant cell wall fibrils
Cellulose polysaccharide microcrystalline fibrils.
497
Plant cell wall fibril cross-linkers
Hemicellulose polysaccharide.
498
Plant cell wall gel
Pectin polysaccharide and lignin polyphenolic polymer.
499
Why is the plant cell wall entirely polysaccharide?
Carbohydrates are the output of photosynthesis, so are readily available for structural support. Proteins regulate the polysaccharides.
500
Cellulsoe fibrils
Polymer of β(1,4) linked D-glucose. Straight, unbranched polymer chain. Most abundant biopolymer on Earth.
501
Microfibril
Unit structure comprised of 18-24 cellulose chains. Inert, micro-crystalline array.
502
How are microfibrils assembled?
Hydroxyl groups allow the cellulsoe polymer chains to hydrogen bond to each other. When lots of the cellulose polymers align, they form a sheet with hydrophilic side surfaces with exposed hydroxyl groups and hydrophobic top and bottom surfaces.
503
How do microfibrils assemble into fibrils?
The hydrophobic surfaces interact and the hydrophilic surfaces interact between microfibrils to form a fibril.
504
Why can't water enter the centre of the microfibril?
It's so densely packed, and all the hydroxyl groups are involve din crystalline bonds. This gives it crystalline properties, which extend to the whole fibril.
505
Cellulose's Crystalinity
Accounts for its insolubility, acid resistance (as acid cannot penetrate the structure) and high tensile strength-- 10^11 N/m^2, similar to steel.
506
Terminal Rosettes
Multimeric enzyme terminal complexes that synthesize cellulose at the surface of the plasma membrane.
507
Cellulose Synthase A (CESA)
Proteins organised into the terminal rosette complex for cellulose synthesis. Display sequence similarity to bacterial cellulose synthase, indicating it was taken up from bacteria.
508
Each CESA protein
Makes one cellulose polymer.
509
Structure of CESA
A transmembrane channel that takes up UDP-glucose from the cytoplasm, cleaves off the UDP then adds the glucose to the cellulose chain.
510
One terminal rosette can produce a cellulose microfibril.
Each rosette has 6 particles. Each particle has 3-6 CESA proteins. One terminal rosette can make 18-24 cellulose polymers.
511
The orientation of rigid cellulose fibres can limit cell growth.
Cellulose fibres are laid down in parallel. The cell will elongate perpendicular to the orientation of the fibrils, in-between the fibrils. Depositing different orientations of cellulsoe fibrils further limits growth.
512
The extracellular matrix is always connected to the cytoskeleton.
The orientation of cellulose fibres is determined by microtubules just under the plasma membrane.
513
How are microtubules orientated?
In parallel, just like the cellulose fibres. Terminal rosettes move along the cortical microtubules.
514
What happens, if there's a mutation affecting the microtubules?
Cellulose fibres are laid down randomly.
515
Why are no motor proteins required to move the terminal rosettes along the microtubules?
Hypothesis: cellulose polymerisation itself provides the energy to propel the terminal rosette forwards, guided by the microtubules.
516
Where are hemicelluloses synthesized?
In the Golgi apparatus, then are transported in secretory vesicles to the plasma membrane with which they fuse.
517
How do hemicellulose cross-linkers form mechanical hotspots?
They branch between cellulsoe fibres to 'glue' them together.
518
Structure of a hemicellulose, Xyloglucan
Side chains lie along one side of a β(1,4) glucose backbone. The other side can interact with cellulose microfibrils.
519
Examples of Hemicelluloses
Xyloglucans, heteromannans, heteroxylans and mixed-linkage glucan.
520
Pectins
Collective name for a group of closely-related acidic polysaccharides. Form an independent gel.
521
What regulates gel consistency?
Ca^2+.
522
Pectins are among the most complex macromolecules in nature.
17 different monosaccharides. >20 different linkages.
523
Where are pectins synthesized?
The Golgi apparatus, then passed to the plasma membrane in secretory vesicles.
524
Properties of Pectins
Hydrophilic. Less hygroscopic than GAGs. More rigid than in animals.
525
Where are pectins found?
In the middle lamella between the two walls. Otherwise, plant cells would move away from each other causing collapse.
526
Functions of Pectins
Influences various cell wall properties including porosity, surface charge, pH and ion balance. Protects against pathogens.
527
What causes growth vecstors?
Turgor pressure from the central vacuole.
528
When can palnt cell growth occur?
When turgor pressure exceeds the yield threshold (which is above the value required to deform the membrane).
529
Recent view of the possible arrangements of polymers in plant cell walls:
Based on structural studies and mutant phenotypes. Some hemicellulsoes separate cellulsoe fibres whilst others join them in mechnaical hotspots. A mobile pectin network loosely cross-links cellulose fibres.
530
The rigid cell wall defines plant cell shape.
Plant cells generally can't move or grow past each other. The shape of organs is determined solely by the growth vectors of individual cells.
531
Daughter plant cells
Conjoined at birth by a shared cell wall. 2 daughter cells originating from the same parent cell will always be next to each other.
532
What is required for plant cell growth?
Carefully regulated loosening of the cell wall. The loosened cell wall means relatively small changes in turgor pressure can have large effects on cell size.
533
Yield Threshold
Above this value, the cell wall yields to pressure.
534
Lockhart Equation
R = ϕ(P - Y), where R is the relative growth rate, ϕ is wall extensibility, P is turgor pressure and Y is yield pressure.
535
What two things need to occur to expand plant cells?
Increase turgor pressure. Increase ϕ by loosening fibril cross-linking.
536
Acid Growth Model
Apopplastic pH is reduced by plant growth hormones, e.g., auxins. Cell walls expand more readily below pH 5. Expansins are activated to disrupt hydrogen bonds by cellulsoe and hemicellulose in mechanical hotspots and reduce pectin rigidity.
537
Primary Cell Wall
In relatively young, growing cells. Flexible, maleable and expandable. Formed between cells after they divide. 90% polysaccharide compared to 65-80% in secondary cell wall. No lignin.
538
Secondary Cell wAll
Develops when the cell reaches its final size. Non-parallel cellulose fibres. Greater rigidity. Multi-layered. Resists biological, physical and chemical attack. 50-80% cellulose (higher than in primary cell wall). No pectin. 15-35% lignin.
539
How do animal cells interact with the extracellular matrix?
Integrins.
540
Integrins
Transmembrane cell-surface receptors that mediate cell adhesion to the extracellular matrix and other cells. Frequently organised into focal adhesions.
541
Integrins form heterodimers
With alpha and beta chains. Depending on the types of alpha and beta chains, they bind to different extracellular components, sometimes via multivalent extracellular matrix proteins, e.g., fibronectins.
542
The extracellular matrix and actin networks…
… are often co-aligned.
543
Focal adhesions form a mechanical linkage between the extracellular matrix and the cell.
This defines cell shape, is essential for cell migration in the trailing edge and is key to signalling (transduces mechanical and chemical information from outside).
544
How do the extracellular matrix and cytoskeleton respond to stress in animals?
Collagen fibres are re-orientated by mechanical stress. Stress fibres re-align to the collagen pattern.
545
How do the extracellular matrix and cytoskeleton respond to stress in plants?
Microtubules re-orientate due to mechanical stress. Orientation of cellulose deposition is altered.
546
What cell types can fibroblasts differentiate into?
Osteoblasts/osteocytes, adipocytes, smooth muscle cells and chondrocytes.
547
The extracellular matrix plays an essential role in fibroblast differentiation.
The cell type into which the fibroblast differentiates depends on where it migrates. If fibroblasts migrate to bone tissue and recognsie components of the extracellular matrix, it will differentiate into a bone cell.
548
Bidirectional Regulation
Between cells and the extarcellular matrix or plant cell wall.
549
Different extracellular matrix components are permissive for neuronal outgrowth.
Some neurones preferentially elongate their axon when on a surface that contains a components of the basal lamina.
550
What initiates new organ formation in plants?
Local cell wall loosening at the apical meristem.
551
WAKs: wall-associated receptor kinases.
Pectin receptors that distinguish between different pectin states. Pectin fragments in repsonse to abiotic and biotic stresses, e.g., pathogens.
552
Gene Isolation Methods
553
Problem: identifying the gene of interest amongst many genes.
Solution: generate a library of many cells/clones that each conatin a different DNA fragemnt. The library can then be screened to identify the cell carrying the gene of interest. Alternatively, use PCR.
554
Problem: to be replicatedin a host, a gene must have a replication origin, i.e., be a replicon, or else it will be diluted during host cell division.
Solution: plasmids and related systems are replicons that can be used as vectors.
555
How can we propagate a DNA fragment of interest?
Introduce it into a vector with a replication origin. Introduce the vectors into E. coli that is grown on agar.
556
Under what circumstances will all the colonies contain the DNA fragment of interest?
If the starting point is the same: all the DNA fragemnts are the same. They are the fragments of interest.
557
What if the starting point is a heterogenous mixture?
The starting point is a mixture containing the DNA fragemnt of interest. Each colony carries a different DNA fragment, so the DNA libraries must be screened to identify the colonies producing the gene of interest.
558
Linn and Arber
Discovered in 1968 that restriction endonucleases were responsible for phage-resistance in bacteria.
559
How do bacteria prevent tehir own DNA from being cut?
Methylases.
560
Why were the enzymes discovered by Linn and Arber unsuitable for gene technology?
They cut randomly-- not a restriction sites.
561
Type II restriction endonucleases.
Discovered in the 1970s. Cut at palindromic recognition sites.
562
Palindromic
Reads the same in both directions.
563
The length of the recognition sequence influences how frequently the endonuclease cuts.
The shorter the sequence, the more frequently the enzyme cuts.
564
Blunt ends
Cutting both strands at the same position.
565
Sticky ends
Cutting both strands in a staggered way.
566
Where does DNA ligase typically come from?
The T4 bacteriophage.
567
Cohesive Termini
Blunt or sticky ends.
568
How long are recognition sites?
Typically 4, 6 or 8 base pairs.
569
EcoRI
Restriction endonuclease from E. coli. R= strain RY13. I= first endonuclease isolated from this species. Other restriction endonucleases incl. BamHI.
570
How is cut DNA joined?
1. Hydrogen bonding between complementary bases, if there are sticky ends. 2. Ligase catalyses phosphodiester bond formation between the DNA backbones in an ATP-dependent reaction.
571
How do we monitor the cutting and joining of DNA?
Gel electrophoresis and intercalating agents.
572
Gel Electrophoresis
Separating DNA fragements on the basis of size. Discovered by Joseph Sambrook.
573
How is gel electrophoresis carried out?
Load the DNA fragment into wells in the agarose gel. The agarose gel immersed in a buffer is placed into an electric field. The negatively-charged DNA fragemnts migrate towards the anode. Smaller = faster.
574
Agarose
Polysaccharide derived from seaweed.
575
Ethidium Bromide
Intercalating agent that inserts itself between the bases of DNA, after which its fluorescence is greatly enhanced.
576
How can we use gel electrophoresis to discern whether DNA fragments have been cut?
Samples are run next to a ladder. Cut fragemnts migrate further and faster than uncut fragments. Ethidium bromide is used to stain fragemnts after gel electrophoresis.
577
When are genomic libraries required?
When interested in the whole transcription unit or intergenic regions.
578
Whole trancription unit
Promoters and introns as well as exons.
579
What do genomic libraries contain (theoretically)?
All the DNA sequences in a cell. Can be >90% non-coding.
580
Why must sample DNA be fragmented before cloning in genomic libraries?
It's not practical to clone entire chromosomes.
581
How is sample DNA fragmented before cloning to create genomic libraries?
Partial restriction enzyme digestion, random shearing or sonication.
582
Why is only partial digestion used?
If the reaction were run to completion, all the restriction sites would be cut, so fragments wouldn't overlap.
583
What does the quality of a genomic library depend upon?
The quality of fragmentation: the maximum number of large, overlapping fragments is required. Fragments are overlapping to ensure no DNA is lost.
584
Why are larger fragments advantageous?
Even if the gene of interest is very small, it is likely to have large intron sequences. Large fragments (~15-20 kb, if using a phage vector) are used.
585
Genomic libarries contain a high number of repetitive sequences
As the library contains everything in the genome.
586
The size of the library required depends on genome size.
If the organism has a large genome, a large genomic library is required to have a good chance of finding the fragment of interest.
587
When is a cDNA library required?
If only interested in the mRNA sequence.
588
Why might a cDNA library not contain all gene sequences?
This depends on the gene expression profile of the sampled tissue as the cDNA library is made from the mRNA of a particular tissue-type of time-point. Contains only exon sequences, e.g., if it's from a brain cell, the same genes will not be expressed as in a liver cell.
589
Why is sample fragmentation not required to make cDNA libraries?
The DNA corresponds to whole mRNA transcripts.
590
What is the extra step required in making this library?
Converting mRNA into cDNA as mRNA cannot be cloned into a vector.
591
Why are insert sizes in cDNA libraries typically small?
~0.5-5 kb as mRNA sequences don't tend to be long.
592
cDNA libraries contain few…
… repetitive sequences.
593
What does the size of the cDNA library depend upon?
Relative abundance of the mRNA of interest. If a gene is expressed at a very low level, a large library is required to have a good chance of obtaining the gene of interest.
594
What does the quality of the cDNA library depend upon?
The quality of the mRNA isolated. Low-quality mRNA is degraded, so won't be in the library.
595
What does the choice of vector for generating either library depend upon?
Size of the genome and size of the gene of interest.
596
cDNA libraries use..
A plasmid or phage as a vector.
597
Genomic libraries use BAC or YAC as a vector.
This is more efficient as the vectors can accommodate the larger fragment sizes required, so fewer clones are necessary to represent the gene of interest.
598
Cosmid
Plasmid with cos sites, enabling packaging into phage particles for more efficient delivery to the host.
599
BAC
Bacterial Artificial Chromsome. Based on the F-plasmid that can carry very large inserts at low copy numbers.
600
YAC
Yeast Artificial Chromosome.
601
What three features are required in plasmid used as vectors?
A selectable marker gene, an origin of replication and a multiple cloning site/polylinker.
602
Selectable marker gene
Encodes ampicillin resistance, so can be used to select the cells that contain the plasmid vector.
603
Multiple Cloning site/Polylinker
Collection of unique restriction sites where the gen of interest is inserted. E.g., in the 5' region of the lacZ gene that encodes beta-galactosidase.
604
If you have successfully introduced the gene of interrest, what will you observe?
Colonies of interest stay white as lacZ is inactivated, so there's no beta-galactosidase to convert X-gal (colourless substrate) into the blue product.
605
Phage lambda has a 48-kb genome.
20 kb is a dispensable stuffer fragment containing genes required for lysogeny. The stuffer fragment is cut out by restriction enzymes, and is replaced by the gene of interest.
606
The recombinant phage infects bacteria.
If the dilution factor is correct, localised cycle of infection, lysis and reinfection give rise to plaques.
607
Phage lambda has cos istes:
12-bp cohesive ends that enable circularisation and packaging in the host.
608
YAC has features of a eukaryotic chromosome.
Telomeres at each end and a centromere.
609
YAC has selectable markers on each chromosome arm.
trp1/ura3 (auxotrophic) and amp^r.
610
YAC Origin of Replication
pMB1 ORI.
611
YAC Cloning Site
sup4 ochre.
612
What does each phage plaque correspond to?
A single clone. Each clone carries a single fragment of genomic DNA from the genome of interest.
613
Preparing a genomic library in a phage:
A restriction enzyme breaks up the chromosome in a partial digestion reaction. The same restriction enzyme is used to cut the vector, so the cohesive termini are compatible. The stuffer fragment is discarded. The phage heads infect the bacteria. Plaques form, if the dilution factor is correct.
614
Synthesis of the first strand of cDNA:
Oligo(dT) primers anneal by heating then cooling. Nucleotides are added. Incubation of the reaction. Reverse transcriptase converts mRNA to cDNA.
615
Why are oligo(dT) primers used?
It binds to the poly(A) tail.
616
Synthesis of the second strand of cDNA:
RNase H nicks the original mRNA strand. These RNA fragments act as primers for cDNA synthesis by DNA polymerase I. DNA polymerase I also has exonuclease activity, so as it adds DNA nucleotides, it also degrades the RNA, so only DNA fragments are left. The T4 ligase joins the DNA fragments.
617
What two things doe screening the library require?
1. Replica of the library. 2. A probe: labelled DNA fragment.
618
Why is a replica of the library required for screening?
To have something that is easy to work with during screening, and to enable preservation of the master plate in a viable form to identify colonies for future propagation.
619
How is a replica plate made?
Overlay a nitrocellulose disc onto the agar of the master plate. Cells stick to the plate. Lyse the cells with NaOH, which also denatures the DNA. Treat the DNA with UV light or bake it to fix it onto the nitrocellulose membrane.
620
Labelling a DNA fragment to make a probe via the random-priming method:
Denature the DNA, then anneal the primer by heating to 95°C then cooling. Add the Klneow fragemnt of DNA Polymerase I and labelled nucleotides. Incubate the reagents. The label generates new strands that are complementary to the template strand, and accumulates the label in the process. Denature to obtain a single starnd to be used as a probe.
621
Template fragment used to make a probe
A fragment of purified DNA similar to the gene of interest. It could be a fragment of the gen of interest, or a similar gene from another organism, for example.
622
Klenow Fragment
Fragment of DNA polymerase I used to label the template DNA because it doesn't have the exonuclease activity of DNA polymerase I.
623
Primer used to make the probe
Random hexanucleotide that can be used to anneal to any fragment you choose. Generic tool.
624
The nucleotides are labelled.
Enables detection of the probe's distribution. The label could be fluorescent, radioactive or with a tag for enzyme-linked detection.
625
Screening the library by hybridisation.
The probe is applied to the replica plate in a buffer, and sticks only to the gene of interest. Wash away any unhybridised probe.
626
Autoradiography
Detecting the radioactively-labelled probe on X-ray film. The probe appears as a black spot. The autoradiograph can be related to the master plate to identify the clone of interest, which can then be propagated.
627
Taq Polymerase
Comes from the thermophilic Thermus aquaticus.
628
Pros of PCR
Library screening can take weeks, whereas PCR is quick and doesn't require a host.
629
30 PCR Cycles
Generates over 1 billion copies!
630
What is required for PCR?
Template DNA, 2x primers (single-stranded oligonucleotides), 4 x dNTPs, DNA polymerase and a thermal cycler.
631
Why are PCR products often cloned in vectors afterwards?
For further manipulation.
632
To design PCR primers…
…the sequence of the target fragment must be known.
633
How can we tell, if PCR has replicated a deletion?
This can be demonstarted by gel electrophoresis as the mutated copies will be shorter.
634
635
Characterisation of Cloned Genes
636
637
How is the gene of interest isolated from bacteria?
Alkaline lysis protocol: SDS under alkaline conditions/NaOH lyses the bacterial cells and denatures the DNA. Add acid to cause the single strands to aggregate. Plasmids are then extracted by centrifugation.
638
How can plasmids be extracted from the rest of the DNA by centrifugation?
The plasmid DNA is supercoiled, so doesn't denature and is found in the supernatant.
639
Comparison of cDNA and genomic sequences
Enables positions of the promoters, introns and exons to be determined.
640
Small gene family
Up to 10 copies.
641
Large gene family
UP to 100 copies.
642
Dideoxynucleotides
Have no 3' OH-- the point at which the next nucleotide in the chain would be added. Block chain extension.
643
Why are the dideoxynucleotides added at non-saturating concentrations in Sanger sequencing?
so that chains do not always terminate at the first position of that nucleotide. The aim is to have a chain that has terminated at every possible position for each nculeotide.
644
Sanger Sequencing: the Dideoxy Termination Method.
1. Mix the template to be sequenced, DNA polymerase, 4 standard nucleotides and 1 dideoxynucleotide at non-saturating concentration. 2. Resolve the products by gel electrophoresis. 3. Automated sequencing: each nucleotide has a different distinguishable tag that is recorded by a laser-based detection system as teh nucleotides exit the tube.
645
Why can Sanger sequencing only be used to sequence up to 800-900 nucleotides?
It's difficult to resolve a 1 nucleotide size difference in a molecule that is already very long. Gradually, the quality of the sequence deteriorates, so the later peaks are well resolved.
646
Why is it more convenient to use fluorescent atgs than autoradiography?
4 different tags can be used, so it can all take place in one tube.
647
What is Sanger sequencing typically used for?
Small projects as it's quick, accurate and cost-effective.
648
Why can't gel electrophoresis ALONE be used to estimate the number of gene copies?
The DNA fragments are so large, they wouldn't move.
649
What is Southern blot analysis used for?
The number of sites in a genome that have similarity to the DNA of interest, i.e., the number of copies of the target gene.
650
What does not need to be known for Southern blotting?
The sequence for the gene of interest.
651
Southern Blotting
1. Genomic DNA is cut with a restriction enzyme. 2. DNA fragments are separated by agarose gel electrophoresis. 3. Capillary blotting: after denaturation with NaOH, separated DNA fragments are blotted onto nitrocellulose paper. 4. Remove the nitrocellulose paper from the agraose gel then fix the DNA to the nitrocellulsoe by baking and/or UV. 5. Hybridisation of teh labelled DNA probe.
652
In capillary blotting, how is the DNA transferred onto the nitrocellulsoe?
Buffer runs through the gel, nitrocellulsoe and inyo paper towels. The buffer draws out the DNA and transfers it onto the nitrocellulose.
653
Making a probe for Southern blotting using a plasmid
Make a recombinant plasmid with the gene of interest. Cut the plasmid with a restriction enzyme to release the gene of interest from the vector backbone. Run the restriction digestion on the agarose gel to resolve the DNA of interest from the vector DNA. Cut the relevant band out of the gel, purify it and label it using the Klenow fragment method.
654
Making a probe for Southern blotting using PCR
Run PCR to generate the gene of interest. Use this is as a template in the method discussed in the previous lecture with the Klenow fragment.
655
How does Southern blotting indicate the copy number?
Count the number of bands. A weaker band may indicate a second divergent version of a gene that's similar to the gene of interest.
656
Purpose of FISH: fluorescent in situ hybridisation.
Determines not only the number of copies of the gen of interest in the genome, but also rough chromosomal locations.
657
Steps in FISH
1. Drop cells of interest onto a glass slide. 2. The cells are fixed and permeabilised, so DNA can enter the cells. DNA is denatured. 3. A fluorescent probe hybridises to the chromosomes. 4. Visualise by fluorescence microscopy.
658
Why are metaphase typically used in FISH?
To enable visualisation of indivdiual, condensed chromosomes.
659
Why is a fluorescent probe preferred to a radioactive one in FISH?
A radioactive probe may not provide sufficient resolution, and may damage the chromosomes.
660
Function of Northern blotting
Analysis of gene expression patterns: when and where is a gene expressed. Provides information on mRNA levels in different tissues and on mRNA size.
661
Why is a fragmentation step not required in Northen Blotting?
The aim is to work with intact mRNAs.
662
Northern Blotting
Resolve the purified mRNA by size using gel electrophoresis. The resolve fragments are transferred to the nitrocellulose membrane by capillary blotting. Hybridise the blot with a probe. Wash away unhybridised probe. Detect the distribution of the probe on the mRNA to determine where on the membrane the mRNA of interest resides. Determine the size of the mRNA transcript by comparison to a ladder.
663
Northern blotting uses a similar principle to…
..southern blotting, but analyses RNA not DNA, and there's no fragmentation step.
664
Reverse-Transcriptase PCR (RT-PCR)
Isolation of the target mRNA. First strand cDNA synthesis. Amplify the cDNA by PCR. Analysis by gel elctrophoresis.
665
Northern blotting vs. RT-PCR
Faster and easier than Northern blotting, but doesn't so easily give information on size comapred to Northern blotting.
666
In situ hybridisation
Determines exactly which cells accumulate the mRNA of interest.
667
Why must a single-stranded probe be made for in situ hybridisation?
A double-stranded probe would re-nature after denaturation, reducing sensitivity.
668
In situ hybridisation: method
1. Fixation of the tissue to ensure the RNA stays in position throughout the procedure. 2. Generate 10 micron-thick sections, so the probe can access the cells. 3. Make a single-stranded RNA probe labelled with digoxygenin. 4. Hybridise the probe to the sample. 5. The DIG tag is detected using an anti-DIG antibody with alkaline phosphotase enzyme conjugated to it.
669
How is the DIG tag detected?
Alkaline phosphotase converts a chromogenic substrate into a insoluble purple precipitate that can be visualised under a microscope.
670
How can a single-stranded, DIG-labelled RNA probe be made for in situ hybridisation?
A plasmid vector carries the cDNA of interest in inverted orientation behind a T7 promoter. T7 polymerase from bacteriphage T7 creates an anti-sense RNA probe that hybridises to the sense mRNA in the sample. The reaction occurs in the prescence of a labelled nucleotide (UTP-DIG) that is incorporated into the probe during synthesis.
671
What is the name for when a fluorescent albel is used instaed of DIG?
RNA FISH.
672
Digioxygenin
A rare plant steroid molecule that is highly antigenic. Widely used as an immuno tag. In in situ hybridisation, it is attached to a UTP nucleotide.
673
Protein Accumulation Profiles
Post-transcriptional regulation means mRNA may accumulate, but the protein doesn't. Where and when does the protein accumulate?
674
How can protein accumulation profiles be investigated?
Western blotting, or in situ tissue sections by immunohistochemistry.
675
A dual-antibody detection system
Used to detect protein. Allows for signal amplification (improving sensitivity) and a single detection reagent (i.e., the secondary antibody conjugate) for multiple protein targets.
676
Primary Antibody
Specific to the protein of interest.
677
How is the primary antibody detected?
Using a secondary antibody conjugated to alkaline phosphatase.
678
How is the primary antibody made?
1. Make the protein in bacteria using a protein expression vector. 2. Wait for the bacteria to grow, then turn on expression of the protein by adding IPTG. 3. Lyse the bacteria to extract the protein. 4. Purify the protein. 5. Immunise an animal, so it produces protein-specific antibodies.
679
What does the protein expression include?
A T7 promoter, shine Dalgarno sequence, a polylinker region with restriction sites into which the target protein's gene can be inserted and a 6-His tag (a sequence encoding 6 consecutive histidines that eventually become fused with the target protein). Origin of replication and a selectable marker.
680
Why can't the target protein be expressed constitutively in bacteria?
It's probably toxic to bacteria.
681
IPTG
Non-metabolisable lactose analogue.
682
How is expression of the protein of interest turned on in bacteria?
There is a lac operator sequence (lacO) between the promoter and the gene of interest in the protein expression vector. The lacI repressor binds to the lac operator sequence. Then IPTG is added, so IPTG binds to the repressor protein, so the lacO no longer binds to the lac operator sequence, and the gene of interest can be transcribed.
683
How is the protein of interest purified?
The 6-His tag binds to the nickel in the beads in the column. Everything else flows through. The protein with the hexahistidine tag can then be eluted.
684
Western Blot Analysis
Use polyacrylamide gel electrophoresis. Use electroblotting to blot proteins to the membrane. Probe with the primary antibody then the secondary antibody then add a chromogenic substrate. By reference to ladder, protein size can be determined.
685
Why does Western blotting use polyacrylamide gel instead of agarose gel?
It provides better resolution.
686
Why does Western blotting use electroblotting instead of capillary blotting?
The polyacrylamide matrix is much tighter than the agarose gel matrix, so extra power is required to move proteins through it.
687
Western blotting can also be used to assess relative abundance.
Relative abundance is proportional to protein size and band intensity.
688
What can be used to analyse protein localisation patterns in situ?
Immunohistochemistry, which uses tissues sections instead of a gel blot, but is otherwise similar to Western blotting.
689
What does comparison of immunohistochemistry with in situ hybridisation illustrate?
mRNA and protein accumulations don't always correlate.
690
691
Organisation of Eukaryotic Genomes
692
693
Maternal Inheritance of Plastids and Mitochondria
Plastid and mitochondrail genomes are in the cytoplasm, and the cytoplasm comes from the female gamete as it's larger, but sometimes there are exceptions where the plastid and mitochondria come from the male gamete.
694
Some cells endoreduplicate.
They copy their DNA, but don't divide.
695
Nuclear Genome
100 Mb- 100 Gb. In several linear chromosomes. 25k-100k genes. 2-64 per cell. Biparentally inherited.
696
Mitochondrial Genome
Bigger than plastid genomes, but with fewer genes. 0.2-2 Mb. Circular. 30-40 genes. 10^2-10^3 per cell. Maternally inherited.
697
Plastid Genomes
10-170 kb. Circular. ~170 genes. <100 per cell. Maternally inherited.
698
3 parent baby
The first was born in 2016. Mother with a mutated mitochondrial genome has her and the male's DNA fertilised in IVF then put into a donor's egg.
699
Smallest Eukaryotic Genome
2.3 Mbp. Parasitic Encephalitozoon intestinalis.
700
Largest Eukaryotic Genome
New Caledonia fork fern. 160 Gb.
701
Genome Size
Usually means the haploid size. Measured by nuclear fluorescence after DAPI staining (C-value), or by nucleotides in a completed genome. DAPI intercalates into dsDNA. When it binds, it fluoresces.
702
C-Value
Size of the genome.
703
For plants and animals, there is only a weak correlation between…
… genome size and number of genes.
704
Parasites tend to have…
…streamlined genomes.
705
Gene Length
At least 3x as long as protein length. ~500-10,000 bp.
706
How many protein-coding genes in most eukaryotes?
10,000-40,000. Higher in polyploid species, but lower in parasites.
707
Longest Eukaryotic Gene
Human Dystrophin 2.3 Mb.
708
Protein Length
~100-1500 amino acids.
709
Longest Eukaryotic Protein
Prymnesium parvum PKZILLA 45,000 amino acids.
710
Gene
Includes everything that is transcribed, so introns and UTRs as well as exons.
711
Purpose of UTRs
Regulatory. Unlike introns, they are part of the mature mRNA, but are not translated into proteins.
712
Gene Organisation
Genes can be in various orientations, and sometimes, uncommonly, they overlap.
713
Genome Browsers
Boxes= exons, lines= introns. Empty boxes= 5' and 3' UTRs. Filled boxes= coding sequence. Only one strand is used as the other strand is redundant. The strand is chosen arbitrarily.
714
Genome Repetitive Content
Can be tandem arrays (telomeres and satellites) or dispersed repeats (transposons).
715
4 of the rRNAs in the ribosome
Comprise 80% of the RNA in a cell.
716
What produces rRNA?
Pol I (45S) and Pol III (5S).
717
5S gene for rRNA
Produced as its own gene by Pol III.
718
45S gene for rRNA
~13 kb is transcribed. The intergenic spacers (non-transcribed spacers) are larger. Arranged in a multiple tandem repeat array with the transcribed and non-transcribed sections repeated. The array can be several Mbp. Produces a polycistronic transcript. This is split up in the cell into the 18s, 5.8S and 28S rRNA.
719
ITS
Internal Transcribed Spacer. So well-conserved in the 45S array, that primers can be designed for it.
720
ETS
External Transcribed Spacer
721
NTS
Non-Transcribed/Intergenic Spacer. Changes, so the primers for the ITS can be used to sequence the spacer. This sequence can be compared between organisms to determine their phylogentic relationship.
722
tRNA
A couple hundred tRNAs are typically found in eukaryotic cells, 15% of the RNA in a cell. Transcribed by Pol III.
723
Where does RNA processing occur?
In the nucleolus.
724
snRNA and snoRNA
Small nuclear and small nucleolar RNAs are involved in RNA processing. snRNAs guide ribonuclear proteins for splicing. snoRNAs chemically modify other RNAs. Usually their part of a riboprotein complex. Produced by Pol II or Pol III.
725
Genes for snRNAs and snoRNAs
A well-conserved class of genes across eukaryotes.
726
Telomerase RNA
Essential component of telomerase.
727
Small RNA Precursors
miRNA, siRNA and piRNA. ~21 nucleotides long. Produced by Pol II, or non-canonical polymerases. Post-transcriptional regulation of gene expression.
728
Canonical Polymerases
Pol I, II and III and in plants Pol IV and V.
729
Long Non-coding RNA
>200 nucleotide tarnscripts produced by Pol II. Scattered across the genome, but infrequently expressed. Often tissue specific. Don't code for protein. Usually regulatory. Can be intergenic, anti-sense to coding genes or even from introns.
730
Centromere
Where microtubules bind to separate the chromosomes during mitosis.
731
Monocentric Centromeres
Point centromeres or regional centromeres. The microtubules only bind at one place.
732
How can you tell if the ecntromere is monocentric?
If the chromsomes appear constricted during mitosis.
733
Point Centromeres
Can be very small, if monocentric-- 125 bp in yeast. Can also be very large, e.g., 5 Mbp, 1/4 of the length of that chromosome.
734
Regional Centromeres
100s of bp -- Mbps of AT-rich satellite repeats, often interspersed with transposons.
735
Holocentric Centromeres
Multiple point chromsomes or arrays of centromic repeats scattered across the chromosome.
736
How can you tell, if centromeres are holocentric?
No primary constriction of chromosomes when imaged in mitosis. They stay straight during anaphase.
737
Why are linear chromsomes damaged over time?
They are not as stable as circular chromosomes.
738
End replication problem due to linear chromosomes in eukaryotes:
When DNA replicates, it requires an RNA primer. The rpimer then degrades, so the 5' end of the chromosome is never reached during DNA replication.
739
Telomeres
Caps at the end of chromosomes to combat the end-replication problem
740
Size of Telomeres
3-10 kbp arrays of short TG-rich repeats. Shrink with age.
741
Telomerase RNA
Riboprotein complex containing telomere RNA (~1.5 copies of the telomeric repeat) and reverse transcriptase. Telomerase binds to the repeat, then uses the telomerase RNA as a primeer to extend the DNA. It then moves along to bind to the newly-synthesized DNA.
742
Satellite Repeats
Have no function. Repeat junk in the chromosome. AT-rich, so are lightly less dense.
743
The satellite repeat unit can vary in size.
Mini- and micro-satellites just vary in the size of the repeat unit.
744
Microsatellite variation between individuals…
Useful in DNA fingerprinting in forensics.
745
Where are satellites found?
Large, monocentric chromosomes. Can be 1/3 of some chromosomes! Minisatellites are found near the centromere. Microsatellies are found everywhere.
746
How do satellite repeats expand and contract in size?
Incorrect repeairs, recombination misalignment, or DNA polymerase stuttering when it encounters the short repeats.
747
Transposon
Genomic parasites that copy themselves around the genome.
748
Transposons are resposnibel for genome szie, largely.
Larger genomes are large, because they have more transposons.
749
Transposons are engines of evolution.
They can insert themselves into a gene to create a new allele, or render a protein-coding gene non-functional.
750
Class 1 of Transposable Element: RNA-based
Transcribed into RNA. Copy, paste. Reverse transcribed to DNA that is inserted at a new location. Once inserted, they don't remove themselves. Some resemble retroviruses. Can increase genome size rapidly.
751
Class 2 of Transposable Elements: DNA-based.
Always stay as DNA, but are transcribed. Cut and paste. Excised from one locus, and inserted into a new locus by transposase. Can amplify via repair and segregation in meiosis.
752
How do transposons cause problems with recombination?
They copy themselves around, and disperse throughout the genome. This can cause changes in chromosoem structure by rearrangements and inversions.
753
Transposons are stress-responsive:
A stress shocks the transposons, so they jump around and create variation.
754
755
Genome Sequencing Programs
756
757
Different Types of Chromosome Map
Karyotypic, linkage, physical and sequence maps (increasing resolution from karyotypic to sequence).
758
Whya re lower resolution maps needed?
To obtain higher resolution maps.
759
Karyotypic Map
Microscopic observations of banding in chromosomal spreads.
760
Linkage Map
The frequency of recombination of polymorphic markers on the chromosome. Units: centiMorgan, cM.
761
Physical Map
Shows the distance between markers. Units: bp. The order of bases is not illustrated. In practice, it may be a tiling path of BAC clones.
762
Why might the distance between markers not eb proportional?
If there are areas of suppression or recombination hotspots.
763
Sequence Map
The end goal of all sequence projects: the sequence of bases along the chromosome.
764
Whose genome was sequenced?
70% of the genome came from one person. The identity of the volunteers was unknown. 5-10 times as many samples were collected as actually used. Volunteers came from Buffalo, N.Y. near the lab. Both male and female donors were used.
765
Why do chromsomes have to be broken up into BAC libraries in hierarchial genome sequencing?
Sanger sequencing runs are limited to 1kb.
766
Hierarchial Genome Sequencing
Chromosomes were broken up into BAC libraries. BAC vectors were clones, so they overlapped. A minimal set of overlapping clones was used to create a tiling path. Each BAC was broken up into 2kb fragments stored in a plasmid, so a new library for each BAC was made-- the shotgun approach. Compile teh sequences of the overlapping plasmid subclones into an entire BAC. Compile the sequences of each overlapping BAC to obtain the chromosome sequence.
767
Celera Genomics
Aimed to sequence the genome more quickly than the public project, patent genes and control access. Used shotgun sequencing instead of hierarchial sequencing.
768
Whole Genome shotgun Sequencing
Bypasses the BAC tiling path. Computationally intensive. Shotgun: fragment the entire genome, and clone the pieces into a plasmid vector directly. Sequencing of plasmid clones at random. Computational assembly.
769
Celera Genomics also had access to the public project data. Why?
Because sequences longer than 1kb had to be published within 24 hours.
770
Next Generation Sequencing (NGS)
Increased the speed and throughput of DNA sequencing. The cost of DNA sequencing dramatically outpaced Moore's Law.
771
Moore's Law
The doubling of computer power and the halving of the cost every 2 years.
772
What was the first NGS on the market?
454 Pyrosequencing in 2005. Removed from the market in 2016.
773
3 Steps in 454 Pyrosequencing
1. Library preparation. 2. Emulsion PCR. 3. Pyrosequencing.
774
Library Preparation
Shear the DNA into 300-800-bp fragments. Ligate olignonucleotide adapters A and B. The fragments are amplified by emulsion PCR instead of being cloned into a vector.
775
Emulsion PCR
Anneal the fragments to an excess of agarose beads that have oligonucleotides complementary to the A/B adapters atatched to them. 1 fragment per bead. Disperse the beads and PCR reagents in oil to form an emulsion with each water droplet carrying a single bead. PCR amplify the unique sequence on teh surface of each bead. Release the beas, and add them to a sequencing plate.
776
Why is it important that each water droplet only carries one bead?
Each droplet functions as a discrete microreactor, eliminating cross-talk during PCR.
777
Sequencing Plate
1.6 million wells. 1 bead per well.
778
Pyrosequencing
Smaller enzyme beads are added to each well to surround the DNA-carrying beads. Sequencing primer, DNA polymerase, APS and luciferin are added. Different dNTPs are added sequentially to the wells in repeated cycles. Incorporation of each nucleotide results in light emission, the intensity of which is recorded.
779
What do enzyme beads contain?
Sulfurylase and luciferase.
780
Why is light emitted when nucleotides are incorporated?
PPi is released, which is converted to ATP by sulfurylase then luciferase uses the ATP to from oxyluciferin + light.
781
Amplification of fragments on the same bead…
…provides sensitivity. It's a massive parallel sequencing approach as there are so many beads involved.
782
454 Pyrosequencing is prone to homopolymer errors.
If there are two or more bases of the same type consecutively, the light intensity is proportionally greater, but this is difficult to measure.
783
Illumina sequencing is reversible terminator sequencing:
It's similar to Sanger, except the terminator can be removed.
784
When did Illumina start?
2006. Now, it has >70% of the market share.
785
3 steps in Illumina Sequencing
1. Library preparation. 2. Bridge amplification: cluster generation. 3. Sequencing.
786
Library Preparation
Similar to 454 pyrosequencing. DNA is fragmented then ligated with oligonucleotide adapters.
787
Bridge amplification-- a type of PCR.
Library fragments hybridise to the oligonucleotides complemnetary to the adapters on the glass flow cell. Amplification is like regular PCR, except all the oligonucleotide primers are atatched to the glass flow cell, so fragemnts must bend into a bridge to bind to the complementary primers. All of the PCR colonies are, thus, constrained in clusters-- polonies.
788
Polonies
Polymerase-generated colonies.
789
Sequencing
The reverse strands are washed away. The sequencing primers anneal. Cluters are supplied with RNA polymerase and all 4 nucleotides. As the nculeotides have their 3' OH blocked, only one is incorporated per cycle.
790
After each incorporation cycle:
The cell is imaged to identify the new nucleotide incorporated at each cluster. Then a chemical step removes the fluorescent tag and 3' OH block.
791
Amplification of fragments on the same bead…
Generates the senitivity required fro detection. Millions of clusters on the glass flow cell can be sequenced simultaneously.
792
How can all nucleotides be added simultaneously instead of sequentially as in 454 pyrosequencing?
Each nucleotide is tagged.
793
2nd Generation Sequencing
454 pyrosequencing and Illumina sequencing. Dephasing is an issue.
794
Dephasing
Once the sequencing of the clonal fragemnts becomes out of sync, then the quality of the sequence data decreases. This limits the lengths of sequencing runs.
795
3rd Generation Technologies
Sequence a single molecule, so dephasing is not issue. Don't involve PCR amplification. Often have high error rates.
796
3rd generation sequencing can be sued in conjunction with Illumina sequencing.
The long reads can be used in sequence assembly.
797
Examples of 3rd Generation Sequencing
Pacific Biosciences and Oxford Nanopore Technology.
798
Resequencing
Sequence a different individual, strain or mutated sequence to identify mutations, polymorphisms etc.. Easier than de novo because new sequence reads can be easily aligned to the previoudly generated genome assembly. Uses shotgun sequencing.
799
What information does RNA-seq provide?
Information on the transcriptome: what genes are expressed, relative transcript levels etc..
800
RNA-seq
mRNA is converted to cDNA by reverse transcriptase. cDNA is sequenced the same way as genomic DNA.
801
The number of RNA-seq seuence raeds of a particular transcript…
… corresponds to the level of expression of that transcript.
802
All sequences in a datbase are annotated with...
Origin, background information, important features within the sequence, e.g., introns, motifs etc. and links to related information such as encoded protein sequence.
803
Issues with Annotation
Annotation is dependent on researchers, so may contain errors.
804
How to annotate:
Identify open reading frames (ORFs). Search databses uses ORFs as queries to identify related genes, conserved functional domains and or protein-targeting sequences.
805
BLAST
Basic Local alignment Search tool, used to find related genes in a database.
806
BLASTN
Nucleotide query search against a nucleotide database.
807
TBLASTX
All 6 different frame translations of a nucleotide query search searched against all 6 different frame translations of a nucleotide database.
808
809
Genetic Manipulation
810
811
Stable Transformation
Introduced DNA is inserted into the host cell's genome, so is replicated as cells divide. Longer-term, more stable expression of the gene of interest. Often non-targeted, but in some species targeted integration is possible through homologous recombination.
812
Transient Transformation
Introduced DNA is not integrated into the host cell's genome, so the target DNA is diluted with every host cell cycle. Suitable for short-term expression, or use in non-rapidly-dividing cells.
813
How else can targeted gene insertion occur?
Gene editing.
814
CaPO4 Precipitation/CaPO4 Transfection
DNA is in a phosphate buffer. The DNA co-precipitates with the CaPO4 when CaCl2 is added. When DNA is added to cells on an agar plate, it adheres to the cell surfaces, and is taken up by endocytosis after 4-16 hours of incubation at 37°C.
815
Pros of CaPO4 Precipitation
Quick, cheap and simple. Not vector-dependent. Can assay for transient expression, or score for stable integration. The latter requires a selectable marker.
816
Cons of CaPO4 Precipitation
Essentially only used for mammalian cell lines.
817
Electroporation
1. Mix the DNA with the cells in an electroporation cuvette. 2. Apply an electric field to the cuvette. 3. This causes ions to move, so the membrane is polarised. Pores form in the cell membrane, so DNA enters. 4. The pores heal.
818
Electric Field
Very short, intense electrical pulse. 2500 V/cm for a few milliseconds for bacteria. Lower for animal and plant cells.
819
Electroporation is a highly versatile method to transform different cell types, but…
… can only be used on plant protoplasts.
820
Protoplasts
Plant cells where the cell wall has been enzymatically removed.
821
Pros of Electroporation
Quick, not vector-dependent, can assay for transient expression, or score for stable integration depending on the system and is used for bacteria, yeast, plant protoplasts and mammalian cells.
822
Cons of Electroporation
Equipment is expensive. If studying a multicellular organism, you need to be able to regenerate from a single cell, as in knockout mice where transformed stem cells are injected into blastocysts which are then implanted into a surrogate mother.
823
When making knock-out or konck-in mice, what is used to target integration?
Homologous recombination.
824
Microinjection
The DNA is injected into the male pro-nculeus before fertilisation. Point of insertion is not controlled.
825
In what organisms is microinjection used?
To create transgenic mice, often. Nematodes, insects, fish, amphibians and mammals.
826
Pros of Microinjection
Not vector-dependent. Allows stable integration albeit usually random.
827
Cons of Microinjection
Expensive due to equipment and animal husbandry. Requires skill. Very labour-intensive.
828
Microprojectile Bombardment
A.k.a. biolistics. Gold or tungsten particles coated with DNA are fired using compressed helium gas as the propellant into tissue in a vacuum.
829
Why is microprojectile bombardment so expensive?
A gene gun can cost £10,000. Cost of gold and tungsten particles.
830
Pros of Microprojectile Bombardment
Quick, not vector-dependent, can in theory be used on any tissue, can assay transient expression, or score for integration and can deliver DNA to organelles (usually chloroplasts).
831
Cons of Microprojectile Bombardment
Equipment is expensive. Best suited for use with robust cells (usually plants).
832
Transplastomic Plasnts
Plants that have modified chloroplats due to the introduction of a transgene into their chloroplast genomes.
833
Why is microprojectile bombardment particularly useful for plant transformation?
Uses force to insert the DNA across the plant cell wall.
834
Viral Vectors for Mammalian Expression
Replication-defective derivatives of natural mammalian viruses. Can be used to deliver DNA to cells or intact organisms (e.g., in gene therapy).
835
Why are adenoviruses so-named?
They were originally found in the adenoids.
836
Adenovirus Vectors
dsDNA. Doesn't integrate into the host genome. Can provoke an immune response with flu-like symptoms. High transgene capacity (>30kb).
837
dsDNA from adenoviruses doesn't integrate into the host genome. What are the consequences of this?
Short-/medium-term expression (weeks/months). Strong transgene expression in non- or slowly-dividing cells. In rapidly-dividing cells, the gene is lost more quickly.
838
How does DNA from adenoviral vectors enter the host nucleus?
The adenovirus binds to the receptor, then is taken into the cell by receptor-mediated endocytosis in an endosome. Acification of the endosome leads to dismantling and release of the capsid. dsDNA enters the nucleus via the nuclear pore.
839
Lentivirus Vectors
Subgroup of retroviruses. E.g., HIV. ssRNA. Integrates into the host genome, which is converted to dsDNA by reverse transcriptase. Allows long-term expression (years).
840
Lentivirus provoke a minimal immune response, but...
… their site of integration is unpredictable, which could cause oncogene activation, and they have limited transgene capacity: ~ 8kb.
841
How are lentiviruses different to other retroviruses?
Can integrate into the genome of non-dividing cells via the nuclear pore, whereas other retroviruses cannot enter via the nuclear pore, so require breakdown of the nuclear envelope, i.e.., cells in mitosis.
842
Pros of Viral Vectors
Effective delivery of DNA to cells in vivor or ex vivo, offer high levels of transgene expression (e.g., adenoviruses), offer long-term, stable integration (e.g., lentiviruses) and are applicable to many different systems due to the diversity of viruses and tehir vector derivatives.
843
Cons of Viral Vectors
Vector-dependent, integrating vectors may activate cellular oncogenes and non-integrating vectors offer less stable expression, particularly in non-dividing cells.
844
Why is the effectivity of viral vectors ex vivo important?
Often in gene therapies, the modifications are made ex vivo then are reintroduced into the patient.
845
Agrobacterium-mediated Plant Transformation
The most common method for transforming plants uses the parasitic bacterium. A. tumefaciens. The activation of vir genes causes the mobilisation of T-DNA that integrates into the plant genome. Hormone and opine genes in the T-DNA region can be replaced with the gene of interest and a selectable marker along with their promoters and terminators.
846
A. tumefaciens
Wounded plants release factors that stimulate the transcription of vir (virulence) genes on the Ti (tumour-inducing, 200 kb) plasmid. This enables bacteria to infect the plant tissue, and tarsnfer the T-DNA region of the Ti plasmid into the plant genome. T-DNA encodes gene for hormones to promote cell proliferation (gall formation, it causes crown gall disease) and opines to nourish the bacteria.
847
T-DNA Region
Transfer-DNA. Can be disarmed and used as a vector. Contains genes for plant hormones (auxins and cytokinins) and opines.
848
Binary Vector Systems
The vir and T-DNA regions are commonly separated into two different plasmids.
849
Pros of Agrobacterium-mediated Plant Transformation
Widely and routinely used. Many vector options are available. Enables stable integration.
850
Cons of Agrobacterium-mediated Plant Transformation
Limited host range: extensive in dicots, but many gymnosperms and monocots are not easily infected. Site of integration is random. Can be time-consuming depending on the species.
851
When transferring DNA, when might the cDNA sequence of an endogenous gene be required?
When seeking to repress endogenous gene expression by RNAi, or to overexpress an endogenous gene with a ocnstitutive promoter.
852
When transferring DNA, when might the cDNA sequence of a relevant gene and a suitable promoter be required?
When expressing a gene in a defined spatial/temporal context.
853
When transferring DNA, when might the sequence of a novel gene be required?
When expressing a novel gene with a constitutive promoter.
854
Where are many constitutive promoters derived from?
Viruses, or commonly expressed native genes such as actin.
855
Promoters for constitutive gene expression in mammalian cells:
SV40 virus, cytomegalovirus, ubquitin and actin.
856
Promoters for constitutive gene expression in Drosophila:
COPIA transposable element and actin.
857
Promoters for constitutive gene expression in yeast:
Alcohol dehydrogenase and cyclins.
858
Promoters for constitutive gene expression in plants:
35S cauliflower mosaic virus, ubiquitin and actin.
859
RNAi: RNA interference.
An anti-viral defence mechanism in eukaryotes, which recognises dsDNA and ca be used to silence a gene/supress gene expression. Even though the target gene is being transcribed, no protein is expressed.
860
RISC
RNA-Induced Silencing Complex.
861
Dicer
Cleaves experimentally delivered dsRNA into ~20-nucleotide-long small interfering RNAs (siRNAS).
862
siRNAs
RISC binds to the siRNAs. The siRNAs guide RISC to the target, complementary mRNA, which it then cleaves.
863
How can the RNAi system be harnessed?
Generate RNAi constructs that can be stably introduced into the genome of the organism of interest.
864
Generate an inverted repeat construct:
The are two sequences corresponding to the target gene, and one is in inverted orientation with an intron spacer in-between them.
865
What is important about the inverted repeat construct?
When the construct is transcribed, it can fold to form a hairpin loop. The intron spacer is spliced out. This forms dsRNA that is processed by Dicer leading to RNAi-mediated silencing of the target gene.
866
Discoverers of CRISPR
Jennifer Doudna and Emmanuelle Charpentier recived the Nobel Prize in Chemistry in 2020.
867
Gene Editing
Uses designer nucleases based around a sequence-specific guiding system that targets the nuclease to the gene of interest. Can be engineered to the desired specificity.
868
ZFN
Zinc-finger nculeases. The guide is protein with a DNA-binding domain.
869
TALEN
Transcription Activator-Like Effector Nuclease. Has a DNA-binding protein domain.
870
CRISPR/Cas9
Clustered Regularly Interspaced short Palindromic Repeat-Associated Nuclease. Uses RNA as a guide.
871
Once in situ, the nuclease creates a double-stranded break with 3 different outcomes:
Non-homologous end joining, or homology-directed repair that leads to either gene correction or transgene insertion.
872
Non-homologous End Joining
If repair is imprecise, there is gene disruption and gene knockout.
873
Homology-directed Repair
If there is a donor template, there is knock-in. Transgene insertion, or gene correction.
874
How does gene editing differ to RNAi?
RNAi is knockdown/partial silencing. Gene editing provides knockout with a very specific point of integration.
875
Polygalacturonase
Enzyme in tomatoes that degrades plant cell walls.
876
RNAi was used to silence polygalacturonase in tomatoes.
Fruit was previosuly harvested early to avoid cracking, but these tasted less flavourful. The RNAi solution meant tomatoes didn't crack when ripe. First sold in 1996. 1.8 million cans sold.
877
Why were the RNAi tomatoes taken off the market in 1999?
The Dr. Arpad Pusztai Affair.
878
Dr. Arpad Pusztai Affair
Lab rats being fed GM potatoes suffered from health issues. An investigation revealed his claims were unsubstantiated, but consumer perceptions had already changed.
879
Glyphosate
Broad-spectrum herbicide that targets EPSP synthase involved in aromatic amino acid synthesis.
880
Round Up Ready Soy Beans
Engineered to express bacterial EPSP synthase, which is glyphosate resistant.
881
By 2020, what % of soy beans in the U.S> were GM?
90%.
882
Why has Europe not accepted GM crops?
Due to safety and environemntal concerns, but they couldn't sustain current livestock levels without soybean imports, which are mostly GM.
883
Virus-resistant Papaya
Expresses PRSV protein coat gene. Interfere with viral replication due to gene silencing. Provides immunity against the virus. Successful in Hawaii. Similar effect to cross protection where resistance is achieved by applying a milder form of the virus.
884
Insect-resistant Cotton expresses the Bt toxin.
Gene encoding the toxin is expressed in plants, so only pests eating the plants are affected by it, unlike spraying which is indiscriminate.
885
Bt Toxin
Used as an insecticide since the 1920s. Derived from the soil bacterium Bacillus thuringiensis. Affects insect digestive systems.
886
Blight-resistant Potatoes
Developed by BASF. German company that could not get license in Europe for 'Fortuna potatoes', so they are grown in the U.S.. Used two genes from the Mexican wild potato that give good resistance to late blight.
887
How many children die due lack of Vitamin A each year?
>1 million, with many more affected by sight loss.
888
Goldren rice produces provitamin A in the grain.
Engineered to produce two genes from the beta-carotene (provitamin A) pathway in grain. Opposed by Greenpeace. Field trails have been disrupted. Still not in widespread use today.
889
Timeline of Golden Rice
Originally developed by Ingo Potrykus in the 1990s, then later improved. Approved for human consumption (2019) and commercial growth (2021) in the Phillipines, then blocked in 2024.
890
Insulin is produced by transformed bacteria.
For treatment of diabetes. Isolated from apig pancreas, but some patients made antibodies against the pig form. Now, the human form is used that is produced by bacteria.
891
Human growth hormone is produced by transformed bacteria.
Treats pituitary dwarfism. Used to be isolated from the pituitary glands of cadavers, but some patients contracted infections. Now, it's produced in bacteria.
892
Hepatitis B vaccine is produced by transformed yeast cells.
Vaccination previously involved an attenuated virus, but this has risks of reactivation or contamination with infectious viruses. Vaccination today uses a recombinant coat protein only. The coat protein is produced in yeast.
893
Tissue Plasminogen Activator/Factor VIII is produced by mammalian transformed cells.
tPA is a de-clotting agent used to treat blood clots, e.g., a pulmonary embolism or stroke. Factor VIII is a clotting agent used to treat haemophillia. Both were previously isolated from blood, but risk of infections, e.g., HIV, were great. Cannot be made in bacteria as complex modifications are required.
894
Mammalian cell cultures are expensive. What are more cost-effective solutions?
Using lactating animals to produce proteins in milk, which are then purified. Or it can be produced in hen eggs.
895
Severe Combined Immunodeficiency, SCID.
Compromised immune system due to a lack of functional T and B cells. The 2 most common types have resulted from single-gene defects (IL2RG (X-linked) or ADA).'Bubble boy' syndrome after, David Vetter lived for 12 years in a plastic, pathogen-free bubble.
896
Therapy for SCID
Deliver the normal gene to the patient's T-cells or hematopoietic stem cells ex vivo using gamma-retroviral vectors. GSK therapy for the ADA-type approved in the E.U. in 2016-- 'Strimvelis'. Retroviral vectors in some X-SCID cases induce oncogene activation leading to leukaemia. Lentivirus vectors and gene editing approaches are being developed.
897
Cystic Fibrosis
Primarily affects the lungs. Patients produce a thick, viscous mucus and are prone to lung infections. Results in a defect in the CFTR (cystic fibrosis transmembrane conductance regulator) ion channel gene.
898
Therapy for Cystic Fibrosis
Deliver the normal gene to the lung epithelia in liposomes in an aerosol from a nebuliser, in vivo. ~4% improvements in lung function were observed. Lentivirus and genome editing strategies are in development.
899
β-thalassemia/Sickle Cell Disease
Patients make less or defective haemoglobin and fewer and defective red blood cells, causing anaemia. Results from defects in β-globin gene.
900
Therapy for β-thalassemia/Sickle Cell Disease
Deliver the slightly modified normal gene into hemapoeitic stem cells from the patient ex vivo using a lentiviral vector (modified HIV)-- LentiGlobin. Many patients in trials have become transfusion-independent. Developed by BluebirdBio, Massachusetts, U.S.. Approved in the EU in 2019.
901
Somatic
Experienced only by the individual receiving the treatment.
902
Germline
The treatment and any side effects would be experiemced by the offspring.
903
Layla Richard
1 year old in 2015. Very ill with leukaemia, so wasn't able to receive the standard treatment of modifying her own immune cells. Instead, immune cells from someone else were modified to evade her immune system. These cells could be used off the shelf for other patients as well.
904
Acute Lymphoblastic Leukaemia
Patients have malignant B lymphoblasts.
905
Bespoke therapy for leukaemia:
Patient's own T cells are engineered via a lentivirus to target cancer/B cells. 90% of patients in remission, but it's expensive and unsuitable for the very sick.
906
Off-the-shelf T cells from another person:
T cells from another person are modified in the same way as in the bespoke therapy, and they are also engineered by TALEN to evade the patients immune system (T cell receptors) and an anti-rejection drug is used.
907
After birth, foetal haemoglobin expression is repressed by the transcription factor BCL11A with adult haemoglobin taking over. How can this be utilised in sickle cell therapy?
Exa-cel or Casgevy: patient's hemapoietic stem cells were modified ex vivo by CRISPR to inactivate the BCL11A gene to increase foetal haemoglobin expression. First patients in trials were functionally cured. Approved as drug in the UK in 2023.
908
909
Heredity, Genetic Linkage and Recombination
910
911
Mendel's 1st Law of Segregation
Each organism posseses two homologous alleles at a single locus. Alleles separate in gamete formation. Alleles separate in equal proportions.
912
Mendel's 2nd Law of Independent Assortment
Alleles at different loci assort independently.
913
Discovery of Chromosomes
1843-- Walther Flemming.
914
Chromosomes were linked to heritable phenotypes.
1901. C. E. McClung et al. identified sex chromosomes in several species. Used insects as they have large, easily-stained cells.
915
Chromosomal Theory
1902. Chromosomes carry hereditary factors. Walter Sutton. Theodor Boveri. Chromosomes segregate independently into chromosomes in gametes. Homologous pairs of chromosomes consist of one maternal and one paternal chromosome.
916
Why did Mendel dismiss the possibility that chromosomes wre responsible for traits?
Peas only have 7 chromosomes, but many more traits. This was resolved by genes.
917
Chromosomal theory and Mendel's 2nd Law appeared incompatible.
Genes located nearby on the same chromosome are said to be linked, and their alleles don't assort independently. The 2nd law should rather state that unlinked loci assort independently.
918
Hemizygote
Possessing a single allele at a locus.
919
Who discovered linkage of nearby genes?
Thomas Hunt Morgan in Drosophila.
920
Sex and eye colour didn't assort independtly in flies. Why?
In the wildtype female, there were two alleles encoding eye colour. In the mutant male, there was just 1 mutant allele on the X chromosome, and nothing on the Y (hemizygote). The gene encoding eye colour was on the X chromosome.
921
How could the hypothesis explaining the linkage between sex and eye colour be tested?
With a test cross. Indicated F1 females were heterozygous, and corroborated the hypothesis.
922
Test Cross
Between a dominant and a homozygous recessive to test the genotype of the dominant individual. The phenotypes of the progeny reveal the genotypes of the other parent.
923
Linkage
Parental combinations of alleles of two or more genes are co-inherited more frequently than predicted by Mendelian inheritance laws.
924
How can you tell, if there's linkage?
In a dihybrid cross between two heterozygotes, assortment of traits doesn't follow the 9:3:3:1 Mendelian phenotypic ratio. In a dihybrid test cross, traits don't follow the 1:1:1:1 phenotypic ratio.
925
Linkage Groups
Groups of genes on the same chromosome display linkage. If A is linked to B and B is linked to c, then a is linked to C etc..
926
Genes in different linkage groups…
… are unlinked, so obey Mendel's 2nd law.
927
Arrangement of Alleles on Linked Genes
Coupling configuartion and replusion configuration. Affects the results of the test cross.
928
Coupling Configuration
Wildtype alleles are on one chromosome, and mutant alleles are on the other chromosome. Dominant alleles on one chromosome. Recessive alleles on the other chromosome. Cis configuration.
929
Repulsion Configuration
Each chromosome carries one wildtype allele and one mutant allele. Trans configuration.
930
Coupling Configuration: Test Cross Results
1:1 WTWT: MutMut.
931
Repuslion Configuration: Test Cross Results
1:1 WT Mut: Mut WT.
932
Why did Hunt Morgan face criticism?
Other people seldom observed his ratios, because they focussed on autosomal linkage rather than sex-linked traits.
933
v and b
Vestigial wings instaed of long. Black body instead of grey. Two autosomal, recessive; linked traits.
934
Alleles assort preferentially in parental combinations, but…
… seldom display absolute linkage as predicted by the linkage hypothesis.
935
What did Frans Alphons Janssenss observe in 1909?
Cross-like shapes made by homologous, non-sister chromatids during meiosis prophase and anaphase.
936
What did T.H. Morgan predict in 1912?
Chromosomes might occasionally recombine, allowing allele combinations to swap. Physical exchange of genetic material between chromatids during cross-over.
937
What happens when crossing over occurs?
Configuration changes.
938
Recombination theory was proven with cytogenetics.
by Barbara McClintock and Harriet Creighton in 1931 using Maize. Chromosome shape changes when DNA is exchanged as physical proof of recombination.
939
Recombination after single strand breaks:
In meiosis, the homologous chromosomes align. Endonuclease cuts. Exchange of DNA across the strands. They are ligated together. Tension in the chromosomes allows the branch point to slide/move. The branched intermediate has to be cleaved to separate the chromosomes. The direction of cleavage dictates the recombination resolution.
940
If cleaved horizontally…
… there is non-recombinant resolution: the recessive alleles are still on the same chromosome, and the dominant allleles are still on the same chromosome.
941
If cleaved vertically…
… there is recombinant resolution. The configuration changes.
942
Holliday Junction
A 4-DNA strand intermediate containing the branch. Due to the tension in the DNA, it resembles a cross. It can rotate to form a flat, branched intermediate. Cleaved by resolvase enzymes.
943
Formation of a Holliday Junction
A D-loop forms between homologous duplexes in which one strand invades the other. Regions of heteroduplex are formed in which complementary DNA from two different duplexes pairs. Both of the linked duplexes contain regions of homoduplex and heteroduplex. Cleavage and ligation results in the unfolded Holliday structure, which interacts with the recombination proteins.
944
There are two axes of symmetry in the Holliday junction: one horizontal and one vertical.
Two of the 4 strands are cleaved by the resolvase enzyme. Horizontal-axis cleavage: the parental strands are still linked-- no recombination. Vertical-axis cleavage: recombination.
945
Recombination after a double-stranded break:
Alignment of homologous chromosomes. A double-stranded break in one of the chromosomes. End resection. Strand invasion then DNA synthesis. Double Holliday junctions. Two resolutions: recombinant or non-recombinant.
946
End Resection
The 3' ends of the DNA are exposed. The other DNA strands can invade and pair up with the ends. The 3' end can be used for DNA synthesis to make a double Holliday junction.
947
Why is suppression of recombination sometimes important?
Keeps certain combinations of alleles linked together. Important in combinations of alleles that determine sex, hence need to be co-inherited.
948
How is recombination often suppressed?
By chromosomal inversions.
949
Recombination can happen in instances of inversions, but…
… results in non-viable chromosomes, so this recombination cannot be inherited as the gametes will be non-viable.
950
Sex chromosomes often have many inversions and extremely low recombination rates.
Hence why Hunt Morgan's X-linked traits displayed complete sex linkage, unlike autosomal traits. No recombination between X and Y chromosomes, but there can still be recombination between X and X chromosomes.
951
How does recombination increase genetic diversity?
It can break linkage, allowing new combinations of alleles to be selected. New mutations can be combined into many more different genotypes.
952
If there is an advantageous allele…
…linked alleles will be inherited with it as the advantageous allele is selected.
953
954
Gene Discovery and Gene Mapping in Eukaryotes
955
956
DNA Polymorphisms
Sequence variations between individuals.
957
Indels
Insertions and deletions.
958
SNPs
Single nucleotide polymorphisms. One nucleotide is different between the two alleles.
959
Forward Genetic Approach
Aims to identify the sequence variation(s) responsible for a particular phenotype. Requires no assumption about the function or nature of the gene product.
960
Reverse Genetic approach
Aims to identify phenotypic changes as a result of sequence variation. Tests a hypothesis about a gene's function.
961
How does the forward genetic approach work?
Isolation of individuals with heritable change in the phenotype of interest due to natural variation or mutagenesis. Identify causative DNA variations.
962
How can random mutations be generated?
UV light or chemical mutagens such as ethyl methan sulfonate (EMS) cause point mutations. X-rays or gamma rays cause large deletions, e.g., of a whole linkage group, incl. several genes. Transposable elements create insertions.
963
Methods to identify mutant genes in eukaryotes:
Insertion mutagenesis, whole genome sequencing or linkage mapping and map-based cloning.
964
Transposons/Transposable Elements
Dsicoovered in maize by Barbara McClintock. Mobile, 'selfish' DNA sequences. Replicate themselves and insert new copies ~randomly in the genome. Can create mutations where they insert into genes.
965
Insertion Mutagenesis
Transposons must be controlled, so aren't active in an organism until induced. The sequence of the transposon is known, so design primers to amplify it as well as a random primer to amplify the flanking sequence. Sequence the fragment produced by PCR, so the sequence of the mutant gene at the point where the transposon was inserted can be identified.
966
Insertion Mutagenesis is of limited use:
Applicable to only a few, well-studied model organisms. Mutation efficiency is low (few mutations). Likely causes loss-of-function mutations.
967
Naturally-occurring polymorphic sequences and induced mutagenesis can create thousands of polymorphic sequences.
E.g., in Drosophila, average EMS concentration induces new mutations at random sites roughly once every 150-300 kb. This is approximately 1 in every 30th gene! Which polymorphic sequences cause the phenotype of interest?
968
When there are thousands of natural or induced polymorphisms, how can the polymorphism responsible for the phenotype be determined?
Linkage is used to identify the position of the gene in the genome.
969
For each pair of genes in a linkage group, there is…
… a different, characteristic recombination frequency. This is due to the distance between them on the genome. The further apart they are, the greater the recombination frequency.
970
When can recombination be detected?
Only in gametes derived from heterozygous parents. Recombination causes a change in configuration.
971
Who hypothesised that recombination for a gene pair depends on their distance apart on the chromosome?
Morgan and Sturtevant, 1911.
972
Recombination Frequency (%) =
Total number of recombination events / Total number of gametes tested x 100.
973
Absolute Recombination Frequency
0
974
Highest Possible Recombination Frequency
50% because the non-parental genotype frequencies sum to 50%.
975
Number of gametes tested is often equal to…
… the number of progeny.
976
How does examining an X-linked trait affecting recombination frequency calculation?
Only take into account F2 females when counting the number of recombinants and the total number of progeny/gametes tested.
977
1% Recombination
1 Map Unit (MU) or 1 centi Morgan (cM).
978
Genetic Linakge Map
Indicates the relative position (order and sitance) or genes in a genome.
979
How is a genetic linkage map generated?
From combining the recombination frequencies for multiple pairs of genes.
980
Below ~30 cM:
Additive distances are similar to measured distances.
981
Above ~30 cM:
Additive distances exceed measured distances.
982
Total additive distance can…
… exceed 50%.
983
As physical distance increases, recombination frequencies are under-estimated.
The further apart two loci are, the greater the probability that more than one recombination event has occurred between them.
984
An even number of recombination events…
… doesn't change the alleles, so is not detected in the chromosome as DNA is swapped then swapped back.
985
Why don't recombination events tend to occur right next to each other?
The crossing over structure takes up lots of space.
986
Genetic and physical maps are colinear (same order of genes), but display poor quantitative correlation.
Genetic map distances often aren't the same as physical map distances due to recombination hot- and cold-spots along the chromosome arms, recombination events rarely occurring next to each other and a lack of recombination near the centromere.
987
The classical genetics approach is time-consuming.
It looks at the phenotype and involves a test cross.
988
Mendel investigated 7 traits. He thought 1 was on each chromosome, but actually…
… it was 7 traits on 6 chromosomes because two of the genes were so far apart.
989
How can linkage mapping be used to identify the position of the gene containing the causative mutation?
Co-segregation of the mutant phenotype with naturally-occurring molecular markers.
990
Molecular Marker
A site of a DNA polymorphism not associated with any observable phenotype, but can be detected with molecular techniques. They serve as reference points because where they are and what alleles they affect are known.
991
When can molecular markers be used?
Only when crossing two individuals with different alleles in homozygous form. The molecular marker is in the same linkage group.
992
How is linkage mapping of a mutant gene carried out?
Generate a mapping population. Identify recombination events between the gene of interest and molecular markers. This gives teh genetic distance and position in teh genome of the mutant gene.
993
How is a mapping population generated?
A homozygous mutant is crossed with a non-related individual with a homozygous wildtype phenotype, which carries a large number of sequence polymorphisms. In the F1 cross, all of the offspring are heterozygous for the molecular marker and the gene of interest. Cross two F1 individuals. 4 F2 combinations form due to recombination and independent assortment. Select recessive homozygotes for the gene of interest to form the mapping population.
994
Why are only F2 recessive homozygotes selected for the mapping population?
These are the only individuals where the genotype is certain based on the phenotype.
995
How can you identify whether recombination has taken place in individuals in the mapping population?
If the allele combination is the same as the parental combination at the molecular marker, no recombination has occurred. Heterozygote at the molecular marker = 1 recombination event. If both alleles at the molecular marker are non-parental, two independent recombination events occurred (one in each gamete).
996
Number of gametes tested =
2 x the number of individuals in the mapping population.
997
Mapping by recombination frequency is probablistic.
Based on estiamtes of frequency, so lots of individuals need to be tested to obtain a reliable estimate.
998
Map-based Cloning/Mapping by Sequencing
Maps the mutation relative to individual recombination events, instead of estimating recombination frequencies.
999
How does mapping by sequencing work?
Make a mapping population the same way as in mapping by recombination frequency. Combine all the recessive homozygous F2 individuals into a bulked mapping population. Sequence the DNA of the mutant parent, the wildtype parent and the bulked mapping population.
1000
How can the output of a whole genome SNP sequencing program be interpreted?
There is 50% recombination between the allele of interest and unlinked SNPs until one position where there is an enrichment of the mutant parent allele at a polymorphism. Frequency here is almost 0-- absolute linkage. You have sequenced the genome, so you knwo where teh polymorphisms are. Thus, the enriched polymorphism and the location of the mutated gene can be identified.
1001
Why is unlikely that the position will be determined exactly in mapping by genome sequencing?
The width of the peak at the enriched polymorphism depends on the number of individuals for mapping.
1002
How can you test whether the enriched allele is responsible for the phenotype?
Causalty is tested with complementation. If it's a recessive mutation, introduce the dominant wildtype sequence into the mutated gene. If the phenotype changes from mutant to wildtype, there is complementation, so the mutation caused the phenotype. No change in phenotype means this mutation was not responsible for the phenotype.
1003
What are the applications of linkage/recombination?
Quantitative trait analysis (GTL), medical genetics, animal and plant domestication, marker-assisted breeeding, selective sweeps and linkage disequilibrium.
1004
1005
Regulation of Transcription in Eukaryotes: Part 1 Transcriptional Regulation
1006
1007
Not all genes are expressed at the same time.
Galactose genes in yeast are only synthesised when galactose is present. Floral induction only after prolonged cold.
1008
Not all genes are expressed in all places.
Haeme biosynthesis genes are only expressed in bone marrow and liver.
1009
WUSCHEL
A gene that regulates how many organs are produced and the size of the palnt. Expressed in the meristem and some cells in the plant embryo.
1010
Tyrosinase
Most people make the same amount of this enzyme, it just has different activity and affects melanin expression. Except in red heads where the tyrosinase gene is less expressed.
1011
KNOX1 Family Genes
Increased expression of KNOX1 genes leads to lobed leaves.
1012
Where is the TATA box?
-35 upstream of the gene.
1013
Assembly of the Pol II Pre-initiation Complex
The TATA-binding protein (TBP) recognises the TATA box. The TBP carries with it TFIID (a 13-subunit complex). This ttriggers the binding of more general transcription factors: TFIIA, TFIIB, TFIIF, TFIIE and TFIIH. The Pol II tail interacts with the general transcription factors, which allow Pol II to associate with the DNA.
1014
No primer is needed for transcription to begin. What must happen instead?
General transcription factors phosphorylate the C-terminal domain of Pol II to begin transcription.
1015
Pol II Transcription
Pol II reads the template strand 3' to 5', so the resulting RNA looks like the coding (not the template) strand.
1016
Where does splicing occur?
In the nucleus.
1017
Features of pre-mRNA
A 5' 7mG gap and a poly A tail, and msut frequently be spliced to remove introns.
1018
Eubacterial systems mostly use…
…repressors.
1019
What is the primary way to regulate transcription in eukaryotes?
Activation. Lack of activation is the basal state.
1020
What % of genes in the eukaryotic genome typically encode sequence-specific transcription factors?
5-10%.
1021
Trancription factors are modular.
Modules are independent, so domains can be swapped and still function.
1022
Transcription Factor Domains
A DNA-binding domain and a functional domain. They can also have dimerisation domains, other protein-protein interaction domians and signal sensing domains.
1023
Functional Domians
Trans-activation or repression domain.
1024
DNA-binding Domains
Highly conserved, so easy to predict from the sequence. Only interacts with a few bases in the DNA. Chromatin structure controls access to these few bases, which is crucial as just a few bases may repeat in the genome. Used to categorise transcription factors.
1025
All domains are encoded on the same peptide.
Domains must be able to fold on their own and interact with a linker region that connects it to something else.
1026
Basic Domains
bZIP and BHLH.
1027
Zinc-Coordinating Domains
Zn finger Proteins.
1028
Helix-Turn-Helix Domains
Homeodomain and Forkhead.
1029
Minor Groove Contacts
MADS Box and HD-ZIP.
1030
DNA-Binding Domain Categories
Basic domains, Zn-coordinating domains, helix-turn-helix domains and minor groove contacts.
1031
Signal Sensing Domains
Can turn the transcription factor on or off, e.g., based on temperature or cell type.
1032
Leucine Zippers
Leucines line up as a way of dimerising.
1033
Homodimer
Two molecules from the same gene. It may be two different transcripts or two of the same transcript read twice.
1034
Heterodimer
Two molecules form different genes. These molecules will still be from the same class, e.g., helix-turn-helix.
1035
Function of Dimerisation
Enables more specificity by binding to a longer sequence and more stable interactions with DNA. Heterodimers also enable more regulation, because each gene encoding a different molecule in the heterodimer may be expressed under different conditions.
1036
Transactivation domains are intrinsically disordered:
They don't fold up into structures, and they lack conserved sequence motifs. They cannot be predicted from the sequence alone and must be determined empirically.
1037
How are transactivation domains determined empirically?
An artifical fragment of the transcription factor of interest is attached to a DNA-binding domain. This is then added to a reproter gene with cis-regulatory sequences to which the DNA-binding domain is complementary. If the reporter is visible, the fragment is the transactivation domian. If it is not visible, it is not the transactivation domain. A similar assay can be used for repressors, but where transcription is the basal state.
1038
How are transactivation domains classified?
By abundant amino acids, e.g., proline-rich, acidic and glutamine-rich.
1039
Repression Domains
Similarly disordered and epmirically identified to transactivation domains.
1040
How do transactivation domains function?
By recruiting common co-activator factors, e.g., Mediator, chromatin-modifying enzymes etc..
1041
Mediator
Large complex that associates with general transcription machinery. Helps to recruit Pol II.
1042
Active Repression
Recruit one of several co-repressor complexes that modify chromatin, or directly inhibit the pre-initiation complex.
1043
Repression by Competitive Interactions
Steric hindrance by binding to the same DNA site (competitive inhibition of the co-activator binding site), forming inactive heterodimers with activators (thus, they couldn't function) and occluding (binding to and blocking) the transactivation domain.
1044
Transcription factors are often the end-point of a signal transduction cascade. What signals affect transcription factor function?
(De-)Phosphorylation, binding of another protein or ligand, nuclear translocation, protein stability, release from a membrane, or another signal such as light, pH or temperature.
1045
Binding of a ligand to a receptor in the cell membrane initiates…
… a kinase cascade that ultimately phosphorylates a transcription factor to activate it.
1046
Co-Folding
When intrinsically disordered domaisn form a structure upon binding.
1047
UAS
Upstream Activator Sequence. A.k.a, a cis-regulatory sequence.
1048
The Trancription Factor GAL4 in Yeast
GAL4 is bound to the cis-regulatory sequence, but its transactivation domain is blocked by the GAL80 repressor, which is bound to it. Galactose binds to and causes a conformational change in GAL3, so GAL3 then binds to and causes a conformational change in GAL80, so that it releases GAL4.
1049
NFkB
A transcription factor involved in the stress repsonse in animals.
1050
How is NFkB regulated?
IkB is an inhibitor that blocks NFkB from entering the nucleus. IkB kinase phosphorylates IkB, as the kinase is activated under stress. Phosphorylation of IkB causes it to dissociate from NFkB, so NFkB can enter the nculeus via the nuclear pore.
1051
Why is notch signalling important to embryonic development?
It indicates to the cell what the neighbouring cells are.
1052
Notch Signalling
Notch is a transmembrane protein with an intra- and an extracellular domain. A ligand on a neighbouring cell binds to the extracellular domain. This attracst proteases that release the Notch Intercellular transactivation Domain (NICD). NCID then translocates to the nucleus, where it forms a heterodimer (by binding to a protein with a DNA-binding domain) to activate gene expression.
1053
Multiple genes are often regulated in parallel by…
…the same transcription factor. A single transcription factor can induce an tire developmental programme.
1054
What happens, if the antennapedia gene is activated?
A cascade is initiated that results in a leg developing from the head instead of antennae.
1055
1056
Chromatin
1057
1058
Human Nucleus Size
10 microns, but has ~2m of DNA inside it.
1059
What type of dye is used to visualise core histone proteins on a gel?
A pH indicator.
1060
Core Histones
10-15 kDas. Positively-charged (20-25% lysine and arginine) to interact with the negatively-charged DNA backbone.
1061
Core histones have a conserved histone fold.
A sequence motif at the C-terminus.
1062
2 copies of each protein in the histone octamer:
H3, H4, H2A and H2B.
1063
How does the histone octamer form?
The H3-H4 dimer is unstable, but two H3-H4 dimers make a tetramer, which is stable. The H2A-H2B dimer is stable, so the tetramer plus two H2A-H2B dimers come together to form an octamer. The H3-H4 tetramer is wound with ~1 loop of DNA before the H2A-H2B dimers associate.
1064
Histone tails extrude beyond the DNA.
These are the unstructured N-termini of the peptides.
1065
~147 bp of DNA
Wraps around the core particle almost twice.
1066
The entry and exit points of DNA…
… are on the same side of the octamer after the DNA has been wrapped around.
1067
H1
Clamps onto the entry and exit sites of the DNA (linker DNA). Not associated with all nucleosomes. Associated with higher levels of chromatin compression.
1068
Linker DNA
~200 nucleotides. DNA near the entry and exit sites. It's not wrapped around a histone.
1069
10nm fibre
Beads on a string/nucleosome array. Insufficient to pack DNA into the nucleus.
1070
The 10nm fibre might fold into the 30nm fibre.
The 30nm fibre causes ~50x compaction of the DNA. Still not quite sufficient for DNA packing.
1071
The structure of the 30nm fibre is unknown.
Solenoid (spiral-like helix of nucleosomes) and zig-zag (has a little twist) have been proposed. It's unclear whether it exists because it hasn't been seen in cells-- only when we isolate chromatin.
1072
10nm and 30nm
Refers to the width of the fibre.
1073
How does the DNA fit into the nucleus?
the 30nm fibre is bound to the nculear matrix to form chromatin loops. The nuclear matrix compacts further and further to form mitotic and meiotic chromosomes.
1074
The exact composition of the nuclear matrix…
… is unknown, but it does contain proteins.
1075
What happens when histones are removed from the chromosomes during metaphase?
The DNA is released in a cloud, forming loops of 30-90kb.
1076
Chromatin loops attached to the nuclear matrix...
… are not all the same size.
1077
What happens to the chromosomes during interphase?
Chromosomes attach to the nuclear matrix, which attaches to the nuclear lamina, and form discrete territories.
1078
What was used to visualise discrete chromosome territories?
FISH!
1079
Heterochromatin
Associated with transposons, and is resistant to gene expression. Parts of the chromatin stained more strongly with the DNA-binding dye.
1080
Where on the chromosome is heterochromatin found?
In the pericentromere (the centromere appears lighter than the surrounding heterochromatin) with occasional knobs of heterochromatin on the distal arms.
1081
Facultative Chromatin
Chromatin only stained darker in certain cell types.
1082
Constitutive Heterochromatin
Chromatin stained darker in all cells.
1083
Euchromatin
Genes are more frequent here.
1084
Mutations cause Drosophila eyes to turn from red to white.
This occurs in the white gene, even though this gene normally encodes red eyes.
1085
Positive Effect Variegation
Variegation: some of the eye is white, but not all. This is caused by X-rays that induce inversions that bring the white gen from euchromatin closer to heterochromatin. The heterochromatin means the white gene was not transcribed. The white gene itself is still functional as the mutation affects chromatin structure.
1086
Additional mutations cause suppression or enhancement of variegation:
Su(var) and E(var) genes.
1087
Su(var)3-64
A histone deacetylase.
1088
Acetylation of histone tails…
… neutralises positive charges on the histone tails, e.g., NH3^+ of lysine can be acetylated. This weakens the ionic interactions with DNA.
1089
Acetylated Histones
Relaxed, transcriptionally active chromatin.
1090
Deactylated Histones
Tight, transcriptionally silent chromain.
1091
HATs
Histone Acetyl Transferases, promote expression.
1092
HDACs
Histone DeACetylases, repress expression.
1093
H3K9 Methyltransferase
H3: histone 3. K9: lysine in the 9th position. A single lysine can be acetylated or methylated, but not both at the same time.
1094
Lysine only has ne NH3^+ in its side chain, so…
… Trimethyl lysine is its maximum methylation.
1095
Methylation
Doesn't intrinsically change the DNA-nucleosome interaction. It is just a chemical marker that can be recognised.
1096
Su(var)2-5
A Heterochromatin Protein 1 (HP1).
1097
Heterochromatin Protein 1 (HP1)
A H3K9me reader. The chromodomain binds to H3K9me. It has a linker domain-- hinge domain. The chromo-shadow domain dimerises, and forms a binding platform for other proteins. The platform only allows effectors to bind when there are multiple, consecutive HP1s. In fission yeast, HP1 recruits an HDAC that condenses the chromatin down.
1098
Su(var)3-3
A H3K4 demethylase.
1099
H3K4me
Methylation of lysine at position for on histone 3 is a defining feature of transcribed genes.
1100
How does H3K4me enable transcription?
By recruiting HATs, chromatin remodelers and general transcription factors.
1101
H3K4me and H3K9me
Mutually exclusive.
1102
Why is heterochromatin found in large blocks by centromeres?
There are loads of transposons here.
1103
Features of Epigenetics
Sequence-independent and self-reinforcing through cell replication.
1104
What happens when DNA is unwound to create a replication bubble?
Histones are evicted, and H3-H4 tetramers are randomly transferred to daughter strands.
1105
When DNA is replicated, new nucleosomes are integrated that must be marked similarly to the pre-replication state. How?
Combinations of chromatin 'reader' and 'writer' domains place old marks on newly-synthesised nucleosomes.
1106
Chromatin remodelling complexes move/remove nucleoosmes in ATP-dependent processes:
Nucleosome sliding, histone exchange and nucleosome eviction.
1107
Pioneer Transcription Factors
Bind to DNA with histones and recruit chromatin modifiers, e.g., HATs, and chromatin remodellers to allow sequence-specific transcription factors to bind for activation.
1108
In repression, what would be recruited to condense the DNA?
H3K9
1109
1110
Gene Regulation in Eukaryotes: Part 2-- Post-transcriptional Regulation
1111
1112
5' Cap and Poly A Tail
Markers on mature mRNA. The 5' cap is placed when RNA Pol II begins. The poly a tail is added after transcription.
1113
Pre-Initiation Complex
40S small ribosomal subunit, charged tRNA with methionine and elongation factors (elF3, elf4G etc.).
1114
How does translation begin?
The PIC recognises the 5' cap, so binds to the 5' end of the transcript. Binding is enhanced by factors associated with the 5' cap. PIC scans the good Kozak context to find the first AUG. The 60S subunit joins to form an intact ribosome and begin translation.
1115
lin-4
The first small RNA discovered. A microRNA in C. elegans that negatively regulates lin-14 without encoding a protein.
1116
What did Viktor Ambros et al. discover in 1933?
Two RNAs from the lin-4 gene: lin-4L (61 nucleotides) and lin-4S (22 nucleotides. Lin-4S is part of lin-4L, and is somewhat anti-sense to the 3' UTR of lin-14, suggesting regulation via anti-sense RNA. This suggestion was demonstrated by Gary Ruvkun et al..
1117
How are miRNAs formed?
The pre-miRNA folds to form a hairpin loop. This is cleaved by Dicer to form a 21-22-nucleotide duplex. The duplex is then bound to the Argonaute protein. One strand (the passenger starnd) of miRNA is ejected and degraded. The strand retained is the miRNA guide strand that guides the Argonaute protein to the transcript.
1118
mRNA Decay
By cleavage or de-adenylation.
1119
Many Argonaute proteins are slicers.
This means they cut one strand of dsRNA, unlike Dicer which cuts both strands. Hence, one strand has a cap but no tail, and the other strand has a tail but no cap. The fragments are degraded by exonucleases.
1120
Deadenylation
Many Argonaute proteins recruit deadenylases. This leads to de-capping and 5' to 3' exonucleolytic decay.
1121
Translational Repression Involving miRNA
Interfering with ribosome scanning (initiation or movement). Argonaute proteins can block the ribosome from moving, or stop the PIC from binding.
1122
miR164 tunes the level of NAC TFs.
miRNAs affect the level of the transcript. It's not necessarily a binary 'on' or 'off'. When miR164 is absent, more of the mRNA is translated, so more serrations on the leaf (encoded by the CUC2 gene, the transcript of which is inhibited by the miR164) occur.
1123
NAC
Family of plant transcription factor proteins.
1124
Synonymous mutations make miRNA-resistant target genes.
In the lab, 4 bases in the CUC2 gene were mutated, so the miR164 couldn't bind, but the redundancy of the genetic code meant the same amino acids were encoded,a nd eth same protein was produced.
1125
What may occur, if the Kozak context is not strong enough?
The ribosome may slide past.
1126
siRNAs
Generally don't have hairpins. An RNA-dependent RNA polymerase makes dsRNA. A different Dicer cleaves it. 2 types. The type produced from a Pol II transcript is used in post-transcriptional gene silencing. The type produced from a Pol IV transcript is used in transcriptional gene silencing via chromatin modification.
1127
piRNAs
Ping-pong mechanism. Slightly larger. ~28 nucleotides. Involved in post-transcriptional and transcriptional gene silencing.
1128
How did the diversity of small RNAs evolve?
Viral resistance. Particularly to RNA viruses that use dsRNA as a replication intermediate. Lots of small RNAs made in infected cells against viruses ate loaded into extracellular vesicles or passed through plasmodesmata in plants to immunise other cells.
1129
Virus-Induced Gene Silencing
A virus can be engineered to produce no symptoms, and a gene is inserted into it. When this infects the cell, small RNAs are produced to silence the inserted gene.
1130
When is virus-induced gene silencing useful?
It can eliminate gene function without mutation. Useful as some organisms cannot be transformed by CRISPR, and some mutations may make the organism inviable.
1131
Why are small RNAs themselves not degraded?
They are bound to the Argonaute protein, and have 2' O-methylation.
1132
RNA cn form many secondary structures.
E.g., the Pseudoknot, the helix, the multi-branch loop and hairpin loops.
1133
G Quadruplex
RNA secondary structure, but not based on base-pairing like other structures. G quadruplexes complex an ion to create a plane. At least three ion complexes form a stacked structure, which is energetically stable. Can be diagonal or lateral.
1134
5' UTR-mediated Gene Regulation
Longer, GC-rich; structured 5' UTRs often have lower translation efficiency and developmental- or tissue-specific expression. If a structure, e.g., a hairpin loop, were very close to the 5' cap, it would sterically block the PIC. elF4a is a helicase that can unwind some structures in the 5' UTR.
1135
G quadruplexes are not constututive.
If a G quadruplex depends on K+ ions, removing K+ ions from the cell causes the G quadruplex to unwind. Other factors could be temperature and/or other molecules.
1136
Interferon gamma (INFG) is regulated via a negative feedback loop.
The pseudoknot in the 5' UTR can be unwound by elF2a, so the interferon is translated. The interferon induces protein kinase R (PKR). Each time PIC passes through the UTR, the pseudoknot reforms. When there's enough PKR, it recognises the pseudoknot, and phosphorylates eF2a, so PIC is repressed.
1137
What is formed when there is too much free iron?
Radical oxygen species.
1138
In the 5' UTR, the protein is only produced in the prescence of free iron.
Under low iron conditions, the Iron Regulatory Protein (IRP) binds to the Iron Response Element (IRE, a hairpin structure), which blocks the PIC, thereby inhibiting translation of metabolic enzymes and iron storage proteins.
1139
What happens in the 5' UTR under high iron conditions?
Iron binds to the IRP, causing a conformational change, so the IRP dissociates from the IRE, and translation occurs.
1140
In the 3' UTR, the protein is only produced in the absence of iron.
Some mRNAs have IREs in their 3' UTRs. Under low iron conditions, IRP bound to the IREs inhibits mRNA cleavage, and enhances translation of iron import proteins.
1141
What happens in the 3' UTR under high iron conditions?
The IRPs can bind to iron, allowing degradation of iron import proteins.
1142
IRPs: recycling and binding.
IRPs can be recycled. Binding is an equilibrium based on affinity.
1143
1144
The Origins and Functions of Mitochondria and Chloroplasts
1145
1146
Mitochondria and chloroplasts are surrounded by double membrane systems.
Outer membrane= permeable. Inner membrane= impermeable. The only other organsisms with double emmbranes are gram negative bacteria.
1147
Internal Membranes
Increase the surface area of the compartment. Cristae and thylakoids.
1148
Functions of Mitochondria
The TCA cycle and oxidative phosphorylation. Ammonia/N-assimilation in glutamine synthesis. Synthesis of branched-chain amino acids (V, L and I) and some lipids.
1149
Functions of Chloroplasts
Photosynthesis and synthesis of most lipids, most amino acids and most vitamins, e.g., beta-carotene (vitamin A).
1150
In eukaryotes, what % of cellular protein is held in mitochondria?
~5%.
1151
In plants, what % of cellular protein is held in the chloroplast?
~70%. ~20-50% of this is rubisco because rubisco is inefficient, so more must be made to compensate.
1152
The vacuole presses chloroplasts out to the periphery of cells.
Hence, chloroplasts are close to the airspaces and there's only a thin layer of cytosol, so the diffusion patwhay for CO2 is minimised.
1153
Why does cytoplasmic streaming move chloroplasts around?
To maximise CO2 acquisition and light absorption. Some cells may be next to other cells, instead of next to airspaces, so chloroplasts must move around to obtain CO2.
1154
Hugo von Mohl
First to describe the 'grains of chlorophyll' in 1837.
1155
Andreas Schimper
Proposed that plants were a symbiotc union of two organisms. First to describe how chloroplasts are propagated by division in 1883.
1156
Konstantin Mereschkowsky
First to propose endosymbiotic theory (that chloroplasts are descendants of bacteria) in 1905.
1157
Margaret Dayhoff
Provided genetic proof that chloroplasts are descended from bacteria in 1978.
1158
Cyanobacteria and chloroplasts look similar under an electron microscope.
Both have large amounts of internal membranes.
1159
Alpha-proteobacteria and mitochondria don't look so similar under an electron microscope.
Alpha-proteobacteria don't have cristae/highly-folded internal membranes.
1160
What do mitochindria in animal cells form?
A highly reticulated, branched; dynamic network with fusion and fission events occurring constantly.
1161
Albert von Koelliker
First to describe mitochondria using a microscope in 1857.
1162
Ivan Wallin
First proposed mitochondria were descendants of bacteria in 1922. Cultured isolated mitochondria in a flask. Big supporter of the hypothesis that symbioses generated new species.
1163
Lynn Margulis
Brought the endosymbiotic origins of mitochondria back to discussion in 1966 with the publication of 'On the Origin of Mitosing Cells'.
1164
Why did it take so long to accept endosymbiotic theory?
It could just be convergent evolution. DNA sequencing has now confirmed the evolutionary origin of the organelles.
1165
Examples of Convergent Evolution
Arms have adapted to wings for flight, eyes for sight, antibiotic resistance when exposed to antibiotics and lactose digestion
1166
Convergent Evolution of Moles
Marsupial mole, Golden mole and European mole all live underground so are blind with a good sense of smell.
1167
Why did domestication of bacteria to form an organelle occur only twice?
Secondary and tertiary endosymbiotic events occur more easily than primary symbioses. E.g., eukaryotic algae have endosymbiosed into other eukaryotes many times. Domesticating a bacterium is more difficult than domesticating a symbiote.
1168
When did endosymbiosis of chloroplasts occur?
At the base of the Archaeplastida.~1 billion years ago.
1169
Inheritance of mictohondri and chloroplasts:
Maternally inherited in almost all eukaryotes. Uniparental inheritance.
1170
When did bacterial photosynthesis evolve?
~3 billion years ago.
1171
When was the endosymbiosis of the mitochondrion?
~2 billion years ago, before the radiation of the eukaryotes.
1172
Why is it difficult to eliminate mutations from organellar genomes?
Organellar genomes are isolated from recombination, so are inherited as a single unit.
1173
Plants dominate biomass on Earth.
Plants comprise 450 Gt of global biomass.
1174
Number of Protein-Coding Genes in Mitochondria
13 in humans, ~35 in plants and 62 in Reclinomonas.
1175
The mitochondrial genome is much smaller than the ancestral alphaproteobacterial genome.
~5000 protein-coding genes in free-living alphaproteobacteria, e.g., Magnetococcus. ~100 protein-coding genes in endosymbiotic alphaproteobacteria, e.g., Rickettsia. 13 protein-coding genes in human mitochondria.
1176
Mitochondrial Genomes in Humans
Circualr genome, 13 protein-coding genes that are all involved in oxidative phosphorylation, rRNA genes (but no ribosomal proteins) and tRNAs.
1177
Mitochondrial Genomes in Plants
The same as in humans, but with extra proteins for cytochrome C biogenesis, ribosomal proteins, maturase and two transporters.
1178
How many protein-coding genes do chloroplasts have?
~80 protein-coding gene in land plants, ~100 protein-coding genes in glaucophytes and ~220 protein-coding genes in red algae.
1179
Chloroplasts have retained more genes than mitochondria.
Chloroplasts have been endosymbionts for 1 billion years, whereas mitochondria have been chloroplasts for 2 billion years.
1180
Reduction of the Chloroplast Genome
~6000 protein-coding genes in free-living cyanobacteria, e.g., Microcystis. ~7,600 protein-coding genes in endosymbiotic cyanobacteria, e.g., Nostoc. ~80 protein-coding genes in the chloroplasts of land plants.
1181
Chloroplast Genomes in Embryophytes
Circular genome, rRNAs, tRNAs, ribosomal protein genes, rubsico large subunit, photosystem genes (PSI and PSII), and genes for complexes I (NADH dehydrogenase-like complex), III (Cytochrome b6f) and V (ATP synthase).
1182
Both chloroplasts and mitochondria have a similar number of proteins.
~3000.
1183
How do so few genes produce so many proteins?
Many of the organellar genes were transferred to the nuclear genome during evolution. Now, proteins are transcribed in the nucleus, translated in the cytosol then imported to the mitochondria or chloroplast.
1184
Why are organellar genes transferred to the nuclear genome?
Uniparental inheritance and Muller's Ratchet. Deleterious mutation tend to accumulate over time (Muller's Ratchet), as the organelles are isoalted from recombination, so are effectively asexually reproducing. The reactive oxygen species generated by ETCs mean there is a high mutagenic pressure in these organelles.
1185
Genome Copy Number per Cell
2 Nuclear genomes, 200 mitochondrial genomes and 5000 chloroplast genomes.
1186
Why are more copies of organellar genes required?
To produce sufficient protein for the cell, whereas only two copies of nuclear genes are required to produce sufficient protein.
1187
Enzymes & the Principles of Catalysis
1188
Why are enzymes necessary?
Biochemical reactions must be specific and fast enough. The active site means substrates are in the right position for the reaction to occur.
1189
Hexokinase
Catalyses glucose --> glucose-6-phosphate. The 1st step in glycolysis. Has beta-pleated sheets & alpha-helices.
1190
Why does hexokinase exclude water from the active site?
To ensure the hydroxyl on the glucose instead of H2O reacts with ATP.
1191
What is the purpose of the Mg^2+ in hexokinase?
To shield some of the negative charge on the ATP phosphate, so that it's more favourable for the OH to react with that particular phosphate.
1192
What happens in the absence of an enzyme?
Product will form & equilibrium is reached given enough time. The more enzyme is added, the faster equilibrium is reached. Rate needs to be fast enough to be compatible with life.
1193
What does triose phosphate isomerase catalyse?
6C fructose 1,6-bisphosphate is converted into 2 x 3C sugars: dihydroxyacetone phosphate & glyceraldehyde 3-phosphate. Only glyceraldehyde-3-phosphate can continue in glycolysis, so TPI converts dihydroxyacetone into glyceraldehyde 3-phosphate.
1194
By how much does triose phosphate isomerase (TPI) increase rate?
From one per day to 4,300 per second!
1195
One of the slowest uncatalysed reactions occurs in the synthesis of the pyrimidine nucleotide UMP.
The uncatalysed rate of orotidine decarboxylation is 1 reaction per 45 million years.
1196
By how much does OMP decarboxylase improve rate?
1 per 39s. A 1.4 x 10^17-fold increase!
1197
Transition State
High-energy, unstable state between the substrate & the product that requires lots of energy to form-- high Ea.
1198
Why is the transition state not an intermediate?
It cannot be captured.
1199
Why is the transition state the least stable most-seldom occupied species in the reaction pathway?
It's the one with the highest free energy.
1200
Enzymes accelerate the reaction…
… by stablising the transition state. This reduces the Ea required to reach it. It's not such a high-energy transition state, so it's easier to form the products.
1201
What remains unaltered by the enzyme?
ΔG & equilibrium position.
1202
Enzymes accelerate the rate of attainment of equilibria…
.. . but don't change the standard equilibrium position (Keq) of the reaction as equilibrium position is a function only of the free energy difference between the reactants & products.
1203
What is true at equilibria, irrespective of the presence of an enzyme?
The rates of the forwards & backwards equilibria are equal.
1204
Before equilibrium is reached…
… enzymes accelerate the rate of the forwards reaction.
1205
Active sites are optimised to stabilise the transition state not the substrate.
The substrate is unstable in the active site, so the Ea for the transition state is lower. The transition site is stabilised, so the product is more likely to form.
1206
What would happen if the active site were specific to the substrate?
The stabilising interactions mean the substrate is unlikely to form the transition state. The Ea to reach the transition stae is even higher than without the enzyme.
1207
Induced Fit
Upon substrate binding, there is a slight change in enzyme conformation, which stablises the transition state.
1208
Catalytic Groups
Key amino acid side chains.
1209
Features shared by enzyme active sites
Crevice in the enzyme structure, catalytic groups are oriented precisely around the bound substrate, some hydrophilic residues are present for substrate binding or catalysis; water is usually excluded unless it's a reactant, so the crevice is lined with mostly hydrophobic amino acids.
1210
Residues involved in binding the substrate…
…usually more extensive than the residues involved in catalysis.
1211
4 Methods of Enzyme Catalysis
Covalent catalysis, general acid-base catalysis, enhancing proximity of reactants & metal ion catalysis.
1212
Covalent Catalysis
Covalent bonds are formed or broken. The active site contains a highly reactive group that becomes temporarily covalently attached to the substrate during catalysis.
1213
General Acid-Base Catalysis
A molecule other than water plays the role of proton donor or acceptor. Usually that molecule is one of the amino acids.
1214
Metal Ion Catalysis
Metals assist via substrate binding, stabilising negative charges on reaction intermediates to facilitate acidity and enabling oxidation reactions. The metal can help widen the scope of reactions as some reactions have too high an activation energy to be catalysed by amino acids alone.
1215
Nucleophile
Species which is strongly attracted to a region of positive charge in another molecule. Has an electron pair available for bonding with an electrophile. They are full negative ions or have a partial negative charge. E.g., hydroxyl, cyanide.
1216
Catalytic Triad
Asp 102, His 57 & Ser 195.
1217
3 enzymes that work similarly to chymotrypsin:
Transpeptidases, acetylcholinesterases & Covid-19 MPro.
1218
Acetylcholinesterase
Breaks ester bonds in the same way chymotrypsin breakd petide bonds.
1219
Transpeptidase
Bacterial serine protease. Important for forming the cell wall. Inhibited by Penicillin, so bacteria are lysed as they cannot form a cell wall when they divide.
1220
Covid-19 Mpro
Uses cysteine instead of serine as a nucleophile. MPro: main chain protease. It's important for the proteins used to form viral packages.
1221
PETases
Found in bacteria in a plastic bottle recycling site in Japan. Enables bacteria to break down plastics to use them as a carbon source.
1222
What do PETases catalyse?
PET is hydrolysed to form MHET (monomeric mon-2-hydroxyethylterephthalate) which is then hydrolysed to form ethylene glycol + terephthalic acid monomers.
1223
PET
Polyethylene terephthalate
1224
Humans are exploiting PETases to recycle plastics.
PETases are being engineered to work under conditions under which the reaction occurs faster. E.g., thermostable, fast & increased binding.
1225
Only 2 things you can measure in enzyme-catalysed reactions:
Formation of product or depeltion of substrate.
1226
Assumptions of the Michaelis-Menten Model
1. Measuring of the initial velocity of reaction, meaning the reaction is not being reversed or near equilibrium. 2. Substrate is in excess of enzyme, so [S]total = [S]free. 3. Reaction is in the steady state, so [ES] is stable.
1227
Km
Stability of the ES complex. Units: M. Usually a measure of the affinity of an enzyme to the substrate.
1228
K3
Otherwise known as Kcat or turnover number. 1st order rate constant. Units: s^-1. A measure of how active an enzyme is in reacting with a substrate once it's formed an ES complex.
1229
2nd Order Rate constant
Kcat/Km Units: M^-1 s^-1. A measure of the catalytic effectivity of an enzyme in dilute solution, good for comapring enzyme activity towards different substrates.
1230
How does the dependence of rate on substrate concentration change?
It declines as substrate concentration increases.
1231
Michaelis-Menten Plot
Plots the initial rates at each substarte concentration against substrate concentration.
1232
V
Initial rate of reaction.
1233
Vmax
Maximum rate of reaction.
1234
How can you find Km?
Halve Vmax.
1235
How can you determine Kcat?
Vmax = Kcat [Etotal] You can find it by finding Vmax, and you know [Etotal] as it's just the total enzyme concentration. All your enzymes wil be saturated at Vmax.
1236
What 2 factors does the 2nd order rate constant take into account?
Turnover efficiency (how good it is at converting enzyme to product) and affinity for the substrate.
1237
Plant Cysteine Oxidases
Involved in plant responses to low oxygen. Used the Km values to measure which enzyme is most sensitive to O2 availability. 5 such enzymes in Arabidopsis.
1238
1239
Charging the Cellular Battery
1240
1241
How far is the reaction displaced from equilibrium in normal cells?
30,000-fold as [ATP] is typically ~3-fold greater than [ADP].
1242
Typical concentrations of ATP, ADP & pi in a cell:
[ATP] = 3 mM, [ADP] = 1 mM & [Pi] = 10 mM.
1243
How much greater is [ATP] than [ADP] in a muscle cell?
Thousands of times greater.
1244
Enzymes in ATP Hydrolysis
ATP is thermodynamically unstable, but kinetically stable. Control when and where ATP is hydrolysed and couple its hdyrolysis to other reactions. Spontaneous hydrolysis of ATP in the cell would take 3-4 hours as it's kinetically stable, so enzymes are required to accelerate the rate of hydrolysis.
1245
What's so special about ATP?
Unusually high equilibrium constant. The intermediate free energy means ATP can also be synthesised easily.
1246
2 Mechanisms Used to Charge the Cellular Battery
Substrate-level phosphorylation & oxidative phosphorylation.
1247
What % of glucose is used by the brain?
0.6
1248
Why is the intermediate free energy of ATP hydrolysis important?
The Gibbs free energy released from ATP hydrolysis is lower than that released by the hydrolysis of other phosphate compounds. The unfavourable reverse reaction (condensation of ADP + Pi) can be driven by the coupling to the hydrolysis of other phosphate compounds.
1249
What molecules can be used to synthesise ATP in glycolysis?
1,3-bisphosphoglycerate & phosphoenolpyruvate. They have greater free energy than ATP, so their hydrolysis can be used to synthesise ATP from ADP + Pi.
1250
Fermentation
Regenerates NAD+ by reducing pyruvate to ethanol or by forming lactate. This allows glycolysis to continue.
1251
Mobile Electron Carriers
Ubiquinone and cytochrome C. Connect the complexes. Not embedded in the membrane.
1252
Why is oxygen the final electron acceptor?
It's easily reduced, highly abundant and forms water, which is benign for the cell. It also has a more positive reduction potential than all the electron carriers, so is willing to accept electrons.
1253
Why does the cytochrome C complex appear red?
The haem pigment rings absorb light. The haem is held in place by the S in cysteine residues or the carboxyl group in other amino acids. Haem groups are used to transfer electrons instead of Fe-S clusters in some complexes.
1254
The more negative reduction potential…
…the more willing an electron carrier is to donate electrons.
1255
The TCA Ccyle
The tricarboxylic acid cycle, the critic acid cycle; the Krebs cycle.
1256
Is life pre-disposed to exploit proton gradients?
Alkaline smokers are a possible crucible for all life on Earth.
1257
Why use oxidative phosphorylation over substrate-level phosphorylation?
Substrate-level is less versatile as it's bound to specific chemistry. In oxidative, lots of different sources of electrons can be used. Electrons can eb extracted then passed to the universal energy carrier-- NAD+. The efficient regeneration of NAD+ also allows the ETC to extract more energy from carbon sources. Oxidative phosphorylation is more efficient.
1258
Why do carboxylic acids play a crucial, universal role in metabolism?
Carboxylic acids were likely the first molecules to spontaneously arise from CO2 + H2. Amino acids and formic acids form spontaneously. Formic acids then condense to form fatty acids. All 3 are carboxylic acids.
1259
The TCA cycle fully oxidises pyruvate.
It starts with a 3C molecule and loses 3 x CO2.
1260
Complex II
Succinate dehydrogenase. Doesn't pump protons or span the membrane.
1261
Fe-S Clusters
Fe has many Oss, so can accept and donate electrons easily. Fe is held in place by S in cysteine residues.
1262
Haem
A tetrapyrrole ring with an Fe atom in the centre.
1263
C ring
The part of ATP synthase embedded in the membrane. The motor, which rotates. 10 c subunits.
1264
How was rotation of ATP synthase demonstrated?
They removed the head and attached actin filaments fused to fluorescent molecules. They isolated the membranes and applied an electric field.
1265
In what direction does the c ring rotate?
Clockwise towards the membrane.
1266
What is the ATP synthase rotary motor made up of?
The proton-conducting unit (the c ring) and the catalytic unit.
1267
Beta Subunits
Where the ADP/ATP nucleotides bind in the catalytic unit.
1268
1269
Metabolism: the Cellular Factory
1270
1271
What is required to add an amino acid to the polypeptide chain?
Hydrolysis of 1 GTP as adding the amino acid is +ve delta G, so unfavourable without coupling to a favourable hydrolysis reaction.
1272
Chains of amylopectin very densely packed
Insoluble
1273
What is required for the addition of each glucose to starch?
2 ATPs: one to phosphorylate glucose and one to add ADP as adenyl-glucose is the monomer that is actually added.
1274
Cellulose
Polysaccharide with beta 1,4 glycosidic bonds.
1275
Where are protons typically sourced from?
Acids and water that dissocaite into H+.
1276
NADPH is ultimately sourced from photosynthesis:
It is used to form GAP that is used to form glucose that is used in respiration asa source of energy for all consumers as you move up the food chain.
1277
Pentose Phosphate Pathway
Used in NADPH and nucleotide biosynthesis. Uses glucose-6-phosphate from glycolysis in the oxidative phase in which it produces CO2 + 2 NADPH. In the non-oxidative phase, ribulose-5-phosphate is converted to ribose-5-phosphate by ribulose-5-phosphate isomerase for use in nucleotide biosynthesis.
1278
NADH
Forms in glycolysis, pyruvate oxidationa and the TCA cycle.
1279
2-oxo-glutarate
Same as alpha-ketoglutarate formed in the TCA cycle.
1280
Glutamatae is used to form alanine by transamination:
The amine from is transferred from glutamate to pyruvic acid to from alanine + 2-oxoglutarate.
1281
Aspartate and Asparaagine form via transamination reactions.
Transaminase catalyses the transfer of an amine group from glutamate to 2-oxoglutarate to form aspartate.
1282
Carbon skeletons for amino acids:
Drawn from the TCA cycle, glycolysis and the pentose phosphate pathway in E. coli.
1283
Tyrosine
Formed from phosphoenolpyruvate and E4P (erythrose-4-phosphate).
1284
Essential Amino acids
Msut be obtained from the diet. Non-essential and conditional can be synthesized by humans.
1285
List of Essential Amino Acids
Histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threonine, tryptophan and valine.
1286
Non-essential amino acids:
Alanine, asparagine, aspartic acid and glutamine.
1287
Conditional Amino Acids
Arginine, cysteine, glutamic acid, tyrosine, glycine, orthonine, proline and serine.
1288
Vitamine B5
Used to synthesize acetyl CoA.
1289
Reactive acetyl thioester bond
Links the acetyl unit to the carbon chain in acetyl CoA.
1290
Fatty acid biosynthesis
Requires acetyl CoA, ATP and NADPH. Acetyl-CoA carboxylase adds an extra C to acetyl-CoA to form malonyl-CoA. Coupled to ATP hydrolysis. A cystein residue in the active site of the FAS has a thiol group that reacts with the malonyl-CoA, so that the malonyl group is attached to teh active site and the CoA is detached. In a reaction involving NADPH, CO2 is lost, and the malonyl group is transferred to the cysteine. This process repeats to extend the carbon chain.
1291
Most complete model of E. coli metabolism:
2251 reactions.
1292
Metabolism is as simple as it can be…
…given biological and biophysical constraints.
1293
High flux pathways
Lots of reactions occurring quickly such as glycolysis and the TCS cycle. Other pathways draw off these.
1294
1. Formation of a doubly-phosphorylated hexose sugar.
2 ATPs are used to diphosphorylate glucose.
1295
2. Generating a doubly-phosphorylated triose sugar.
Splitting the diphosphorylated glucose into 2 triose sugars forms NADH.
1296
3. Substrate-level phosphorylation
Transfer of the phosphoryl group from the triose sugar to ADP to form ATP. ATP generation is the ent result of the pathway.
1297
Glycolysis must generate a range of intermediate molecules…
…that provide carbon skeletons for the biosynthesis of acrbohydrates, amino acids and lipids.
1298
Small, uncharged molecules can diffuse passively across the membrane.
Addition of negatively cahrged phosphates early in the pathway prevents the loss of substrate.
1299
Glycolysis must avoid high-energy intermediates that would be toxic to the cell.
The more efficient formation of glycerate-1,3-phosphate instead of glyceraldehyde-3-phosphate would produce the highly reactive, toxic intermediate methylglyoxyl that react with proteins and DNA in cross-linking reactions that would affect DNA replication and protein function.
