Lectures 20-37 Flashcards
(208 cards)
1
Q
What are the phases of protein development?
A
The burst phase (0-5ms), the intermediate phase (5-100ms) and the final rate limiting step.
2
Q
What is the burst phase?
A
It involves formation of secondary structure and collapse of the hydrophobic core.
3
Q
What is the intermediate phase?
A
It involves formation of a molten globule intermediate, which has characteristics of both folded and unfolded proteins.
4
Q
What is the rate limiting step?
A
The attainment of native structure. The final transition is marked by conversion of the molten globule via global repacking of hydrophobic side chains and the association of domains that were folded independently in the intermediate stages.
5
Q
What is a molecular chaperone?
A
Molecular chaperones are proteins that bind to and stabilize an otherwise unstable conformer of another protein, and facilitate its correct fate in vivo: be it folding, oligomeric assembly, transport to a particular subcellular compartment, or controlled switching between active and inactive conformations.
6
Q
What is the structure of chaperonin?
A
Homo-oligomer of 14 subunits (each of 60 kDa) arranged into 2 stacked rings each of 7 subunits. Each ring is structured like a donut with a 7-fold axis and a chamber.
A smaller lid structure, also comprising 7 subunits, sits on top of one of the barrels. Unfolded proteins (orange) bind to the rim of the barrel and are displaced into the cavity by the lid structure. The protein can then fold in a sequestered and protected environment of the chamber. The lid dissociates due to changes in the conformation of the large subunit as ATP is hydrolyzed.
7
Q
Discuss the function of heat schlock transcription factor.
A
Molecular chaperone gene transcription is controlled by Hsf which responds to the presence of unfolded protein or heat shock or other types of proteotoxic stress.
8
Q
What degrades proteins?
A
Protein degradation in the cytosol and nucleus is largely accomplished by the proteasome, a large gated protease. Proteasome substrates are targeted via covalent linkage to multiple copies of ubiquitin, a 7 kDa protein.
9
Q
What is the structure of the proteasome?
A
The proteasome comprises a central catalytic core (20S proteasome) and a regulatory cap (19S) that together are called the 26S proteasome. The 20S core of eukaryotes is comprised of 2 copies each of 14 different subunits, although these fall into two categories of α-type and ß-type. Access to the channel is via the tunnel formed at the ends of the α-subunit rings. It is thought that a single unfolded polypeptide transits into the proteasome at one end and is degraded progressively in the central chamber.
10
Q
What are the different activities of proteasomes?
A
The three activities of eukaryotic proteasomes are chymotrypsin-like (cleaves after hydrophobic amino acids), trypsin-like (cleaves after basic amino acids), and peptidyl-glutamyl peptide hydrolyzing activity (cleaves after acidic amino acids). Two additional activities have been described in mammalian proteasomes (cleaving after branched chain amino acids and between small neutral amino acids). The products are peptides in the 7-9 amino acid range.
11
Q
What does the 19S regulatory subunit do?
A
The 19S regulatory complex contains subunits that: recognize ubiquitinylated substrates, deubiquitinylate the substrates; and prepare them for proteolysis via protein unfolding if necessary. The 19S complex is comprised of at least 15 subunits, six of which are AAA-ATPases that can perform unfolding.
12
Q
What does ubiquitin do?
A
Ubiquitin is a 76-amino-acid protein that becomes covalently attached to polypeptides
that are substrates for degradation. Ubiquitin is linked in linear chains where the carboxyl end of the terminal glycine becomes covalently attached to the epsilon amino group of lysine 48. The attachment of ubiquitin to proteins requires the action of three different types of enzymes called E1, E2 and E3.
13
Q
What does E1 do?
A
E1 is an enzyme that carries out ATP-dependent activation of the C-terminal glycine in a two-step reaction. First a ubiquitin-adenylate is formed, followed by transfer of activated ubiquitin to a thiol site in E1. The high-energy thiol bond is important for Ub transfer to E2 enzymes. There are only a small number of E1 enzymes.
14
Q
What does E2 do?
A
The E2 enzymes, or ubiquitin conjugating enzymes, accept the ubiquitin from E1 and transfer it to the protein substrate in a reaction that requires E3, the ubiquitin protein ligase.
15
Q
What does E3 do?
A
E3 enzymes, or ubiquitin ligases, are a large and diverse protein family that specifies substrate selection.
16
Q
Draw the ubiquitin/proteasome pathway.
A
See screenshot 8.
17
Q
What does CHIP do?
A
It binds directly to Hsp70 and catalyzes ubiquitinylation of misfiled proteins. Imperative for quality control.
18
Q
What are aggregates?
A
Aggregates occur when misfolded proteins overwhelm the ubiquitin/proteasome pathway. Aggregates represent amorphous assemblies of misfolded proteins bound together via hydrophobic interactions or ordered assemblies of amyloid fibres. Both types of aggregates are inaccessible to the proteasome and must be cleared by the autophagic system.
19
Q
What is the aggregate in Alz and amyloid disease?
A
Amyloid beta extracellularly and hyperphosphorylated tau intracellularly.
20
Q
What is the aggregate in Parkinson’s?
A
Lewy Bodies.
21
Q
What are some polyglutamine repeat diseases?
A
Huntingtons, SMBA, DRPPLA etc. In some cases, the triplet encoding Q (CAG) expands during replication and reaches many more residues than it was supposed to have which can be pathogenic depending on the protein.
22
Q
What are prion diseases?
A
Prions are transmissible amyloids and cause disease in humans and other
mammals. Transmission can occur by eating contaminated food. Human prion diseases include Creutzfeldt-Jakob disease and fatal familial insomnia. All known prion diseases attack the brain.
23
Q
What does the ER do?
A
The Endoplasmic Reticulum is a protein folding and quality control compartment
24
Q
What does the Golgi apparatus do?
A
Golgi Apparatus is important for sorting of proteins towards different parts of the cell.
25
What does the rough ER do?
The rough ER contains ribosomes and is important as a protein- folding compartment.
26
That does the smooth ER do?
The smooth ER contains membrane- bound enzymes important for lipid synthesis and metabolism, as well as detoxifying enzymes such as cytochrome p450s.
27
How are newly made proteins targeted to the ER?
Newly-made proteins are targeted to the ER membrane by an N-terminal signal
peptide. The signal peptide is 8-20 residues and enriched in hydrophobic amino acids, and is often cleaved after import into the ER. Some proteins have internal targeting sequences that are not cleaved after import. The signal peptide binds to the Signal Recognition Particle (SRP), a ribonucleoprotein complex that attaches to newly- synthesized proteins while they are still being translated.
28
What happens to the protein once SRP binds to the N-terminal signal peptide?
Once SRP binds to a signal peptide, translation is arrested and the complex of translating ribosome and SRP bind to the ER membrane via an SRP receptor complex. SRP receptor sits adjacent to the translocation channel called the Translocon. This binding is positioned so that
translation can resume in the context of translocation through the aqueous channel and into the lumen of the ER. For membrane proteins, this channel opens sideways, into the plane of the membrane, and membrane-spanning domains of proteins become inserted into the membrane itself.
29
How does the ER lumen differ from the cytosol?
The ER is an oxidizing environment and therefore distinct from the reducing
environment of the cytosol. The oxidizing environment helps to facilitate folding of proteins that must exist outside of the cell, which is also an oxidizing environment.
30
Proteins that enter the ER lumen use?
Fold in association with molecular chaperones, including an ER-specific form of Hsp70 and many others.
31
What important step for protein folding occurs in the ER lumen?
Proteins entering the ER are also glycosylated on asparagine (N- linked glycosylation) via a 14 residue unit that includes 3 residues of glucose, 9 residues of mannose and 2 residues of N-acetyl gluosoamine (GlcNac). These are transferred from a dolichol anchor to the substrate protein. The three glucose residues have important roles in protein folding.
32
What are the three types of membrane proteins?
Type I= N terminus in the ER lumen
Type II= C terminus in ER lumen
Or, they may be topologically complex and have multiple membrane-spanning domains comprising hydrophobic amino acids that form alpha helices.
33
What is the unfolded protein response (UPR)?
When the amount of unfolded proteins in the ER exceeds the chaperone apparatus to facilitate folding, the UPR signaling pathway is activated. Unfolded proteins activate receptors on the ER membrane that then activate transcription regulators. These travel into the nucleus of the cell and increase the transcription of chaperone molecule mRNA (same with ubiq/prot pathway stuff too). This increased mRNA output is transported back to the ER, is translated with the help of a ribosome and raises the amount of chaperones available to help fold protein in the ER.
34
What is a route other than UPR that allows the ER to avoid the aggregation of misfiled proteins?
Increased expression from the UPR is accompanied by ER-associated degradation (ERAD) where luminal and membrane proteins are retranslated from the ER to the cytosol for degradation by the proteasome.
35
What is the structure of the Golgi apparatus?
The Golgi apparatus comprises a stack of flattened membranous disks that have a distinct curved appearance. The curvature creates polarity with a ‘cis’ face and a ‘trans’ face at the concave end.
36
What is the function of the Golgi apparatus?
The Golgi receives all proteins that leave the ER. Once inside the Golgi, these proteins are modified by post-translation modification, such as trimming of carbohydrates, phosphorylation and sulfation. These modifications increase the complexity of the proteins traveling through the Golgi and also function to specify subsequent localization to other membrane systems.
37
How do proteins get from the ER to the Golgi?
Proteins exit the ER in lipid vesicles that bud from the ER membrane (the formation of vesicles will be described in more detail below). They are targeted to the Golgi where they fuse at the Cis side of the organelle.
38
What happens to ER proteins that accidentally get transported to the golgi.
They are transported back by a retrieval pathway. This pathway uses a receptor that recognizes a special peptide sequence (KDEL) at the C-terminus of resident ER proteins.
39
How are lysosomal enzymes recognized and sequestered for transport?
Lysosomal enzymes are identified in the cis-Golgi by an enzyme that phosphorylates on one of their mannose residues of the core carbohydrate unit that was added in the ER. This phosphorylation is recognized in the trans-Golgi by the mannose-6-phosphate receptor that helps sequester lysosomal enzymes into specific vesicles for transport to lysosomes.
40
Describe the process of endocytosis
Some species, let's say in this case LDL, blinds to its receptor on the cell membrane. The membrane then pinches off forming a vesicle containing both the species and the receptor with a clathrin coat. The vesicle undergoes "uncoating" in the cytosol and then fuses with an endosome. The receptor then buds off from the endosome into a transport vesicle that is transported back to the membrane and the endosome transfers the LDL to a lysosome.
41
What is autophagy?
Autophagy is a mechanism of delivering intracellular components to the
lysosome for destruction and recycling. Organelles or protein aggregates are engulfed by a double membrane system for delivery to the lysosome. In some cases, molecular chaperones such as Hsp70 can directly deliver proteins to the lysosome for destruction.
42
What are the 3 types of coat proteins?
Clathrin, COPI, and COPII
43
Where do COPII-coated vesicles go?
COPII coated vesicles serve transport from the ER to the Golgi.
44
Where do clathrin coated vesicles go?
Clathrin coated vesicles serve transport from the trans-Golgi network to the plasma membrane and also to endosomes.
45
Where do COP-1 coated vesicles go?
They transport proteins from the cis end of the Golgi complex back to the rough endoplasmic reticulum (ER)
46
What is the name of the protein that uncoats clathrins?
Dynamin
47
What are rabs and what do they do?
Small GTP binding proteins that target vesicles to certain membranes using proof reading and interacting with specific tethering proteins on target membranes.
48
Snare proteins work on what two processes?
Vesicle targeting to membranes and vesicle fusion.
49
Why does vesicle fusion need help?
Fusion of a vesicle with a target membrane is energetically unfavorable because
the hydrophilic head groups and their associated water molecules represent a barrier to the mixing of hydrophobic hydrocarbons.
50
How do SNARE proteins work?
The energy barrier for vesicle fusion is overcome by interactions of specific SNARE proteins on the vesicle (v-SNARE) and target membrane (t-SNARE). The SNARE proteins interact with each other in such as way as to bring the vesicle into very close apposition to the target membrane, squeezing out water molecules and reducing the thermodynamic barriers to lipid mixing. Similar mechanisms are employed when viruses enter cells. SNARE proteins are disassembled after fusion with the help of a specific chaperone. They are then recycled to their respective membrane systems.
51
Describe the outer membrane of a mitochondria
The outer membrane is porous to molecules up to 5-10 kDa and contains a translocation apparatus called the TOM complex for translocation of proteins.
52
Describe the inner membrane of the mitochondria.
The inner membrane is 70% protein and impermeable even to protons. It is folded into many christae to increase surface area. The inner membrane contains the protein complexes of the electron transport chain and the ATP synthase complex that catalyzes formation of ATP from ADP. Movement of protons across the inner membrane creates a membrane potential. The inner membrane also contains several channel proteins for the translocation of metabolites (eg. pyruvate, malate, acyl-CoA, amino acids), ions and the ADP/ATP transporter. Finally, the inner membrane is also the site of a translocation complex for proteins entering the mitochondrial matrix.
53
What is in the inter membrane space?
An inter membrane space exists between the two membranes and contains enzymes that phosphorylate other nucleotides apart from ADP eg. nucleoside diphosphate kinase which converts GDP to GTPWhat is inside the mitochondr
54
What's inside the mitochondrial matrix?
Hundreds of enzymes, including those required for oxidation of pyruvate, fatty acids, ketone bodies to acetyl-CoA. The matrix is also home to enzymes that catalyze amino acid oxidation and enzymes of the tricarboxylic acid cycle and urea cycle. The mitochondrial matrix is also contains the mitochondrial genome, ribosomes, tRNAs and molecular chaperones for folding of newly synthesized and newly imported proteins.
55
What is different about the codons in mitochondria?
The mitochondrial genome uses a distinct genetic code. For example, UGA is a universal STOP codon, but in the mammalian mitochondrial genome it encodes Tryptophan. AGG encodes arginine in the universal code but is a STOP codon in the mitochondrial genome.
56
What does the mitochondrial genome contain?
The proteins encoded by the mitochondrial genome are subunits for several components of the respiratory chain including cytochrome c oxidase, NADH dehydrogenase and apocytochrome b. The human mitochondrial genome encodes many of the genes needed for protein synthesis within the matrix including 22 tRNAs as well as 12S and 16S rRNA that are part of the mitochondrial ribosomes.
57
Where are most mitochondrial proteins made?
Most mitochondrial proteins are synthesized on cytosolic ribosomes
before targeting to the outer membrane and post-translational import. Nuclear encoded mitochondrial genes contain a targeting sequence. These are of two types. The first is an 15-35 residue N-terminal sequence comprising basic amino acids that is cleaved in the matrix by an endoprotease. The second type of targeting sequence is a non-cleaved internal sequence.
58
What is the role of HSP70 in the mitochondria?
Hsp70 molecular chaperones play key roles in translocation of proteins into mitochondria on the cytosolic and matrix sides of the membranes because proteins are imported in an unfolded conformation.
59
What mediates import across the mitochondria outer membrane?
The TOM complex. It associates with several import receptors that bind to mitochondrial pre-sequence containing proteins at the outer membrane. These import receptors bring the pre-sequence containing proteins to the translocation channel formed by the protein called TOM40.
60
What mediates transport across the inner mitochondrial membrane?
Transport across the inner membrane is mediated by the TIM complex. The positively charge pre-sequence plays a role in opening the channel in the TIM complex and the membrane potential is important for subsequent translocation by an electrophoretic effect. Hsp70 is also important for translocation of pre-sequence containing proteins into the matrix.
61
Draw the mitochondrial protein translocation.
just google it I'm tired of screenshots.
62
What is a peroxisome?
Small single membrane organelles that get their name from metabolism of hydrogen peroxide in the organelle. Peroxisomes have important roles in fatty acid ß-oxidation. This resembles ß-oxidation in mitochondria except that hydrogen peroxide is the by-product. The hydrogen peroxide is metabolized by the enzyme catalase to water and oxygen.
63
Where are peroxisome proteins coded?
The nucleus then produced in cytosolic ribosomes. They are imported fully formed and folded using peroxisome target signals (PTS)
64
What are two peroxisomal diseases?
Zellwegers syndrome, where there is no import of any peroxisomal enzyme.

Adrenoleukodystrophy (ALD), where oxidation of very long chain fatty acids is defective.
65
Discuss the structure of the nucleus.
The nucleus is surrounded by a double lipid bilayer, the outer one being contiguous with the rough ER. The perinuclear space between the membranes is contiguous with the lumen of the ER.
A distinct structure (10-20 nm diameter) lines the inner surface of the nuclear membrane called the nuclear lamina, comprising a meshwork of intermediate filament type proteins termed lamins (more about lamins in lecture AC7). Transport into and out the nucleus is via aqueous pores called nuclear pore complexes. Inside the nucleus, there are distinct structures that comprise the nucleolus, where rRNA is transcribed and
where ribosomal subunits are assembled.
66
What is the nucleolus?
The nucleolus is the most prominent structure within the nucleus and is the site of rRNA synthesis, processing and ribosome assembly.
67
What does RNA PolymeraseII do?
Transcribes ribosomal protein genes.
68
What happens when ribosomal protein genes are transcribed?
They exit the nucleus and are translated on cytoplasmic ribosomes. The ribosomal proteins then reenter the nucleus and are transported to the nucleolus, as does 5S rRNA, where they assemble with rRNA. Individual 40S and 60S pre-ribosomal particles are then delivered to the cytosol.
69
Nuclear Localization Sequences (NLS)?
An NLS is typically 4-8 amino acids, rich in Arg and Lys and usually contains Pro. Nuclear export signals (NES) exist that are distinct from NLS import signals. NLSs are not removed because proteins shuttle between the nucleus and cytoplasm or have to reenter the nucleus as it reforms after mitosis.
70
What is the general structure of a Nuclear Pore Complex (NPC)?
The NPC is composed of several ring assemblies that occupy the cytoplasmic face, inner core and nucleoplasmic face of the structure. Filamentous assemblies radiate out from both cytoplasmic and nucleoplasmic sides.
71
Draw the nuclear transport mechanism
Screenshot 9
72
What do karyopherins do?
Transport into and out of the nucleus depends on specific transporters, called karyopherins, that recognize the NLS or NES sequences.
73
What is Ran and what does it do?
Ran is a small GTPase with co-factors that regulate nucleotide hydrolysis (Ran GAP) and nucleotide exchange (Ran GEF). Ran GAP exists primarily in the cytosol whereas the Ran GEF is nuclear. Therefore, Ran is in the GDP form in the cytosol and in the GTP form in the nucleus.
74
Describe the structure of Intermediate filaments.
IF monomer is elongated and alpha-helical with a globular N-terminus head and globular C-terminal tail. IF forms a dimer that is a coiled coil.** The dimers associate to form a staggered tetramer. Each of the tetramer forms associates with 7 others to form a filament containing 8 tetramers. IFs have no polarity because the tetramers form head to tail.
75
What do IF's do?
Ifs have rope-like character – easily bent but not broken. Ifs form network in cell and often surround nucleus, forming a network towards the cell periphery. They interact with junction proteins called desmosomes or hemidesmosomes.
76
Name the four classes of IF's.
Keratins, Vimentins, Nucleofilaments and Nuclear Lamins
77
What IF's assembly is reversible and why?
Most intermediate filaments do not have reversible assembly with the exception of nuclear lamins. In this case, assembly is reversible due to phosphorylation that is cell cycle dependent. This allows for nuclear breakdown during mitosis. Mutations in lamin proteins lead to premature ageing.
78
What are microtubules?
Long hollow tubes that are very dynamic. They function in intracellular organization and intracellular transport. They form the mitotic spindle and also cilia and flagella. Cellular microtubules (MTs) grow out of the centrosome.
79
What is the structure of microtubules?
MTs grow from a tubulin dimer of α-tubulin and ß-tubulin. Both bind GTP. MTs form a linear protofilament that organizes laterally into a tube containing 13 protofilaments.
MTs have polarity because dimers prefer to bind to exposed ß-tubulin surface rather than α-tubulin in a protofilament. Exposed ß-tubulin surface is the plus end while the exposed α-tubulin is the minus end. In cells, the MTs grow out of the centrosome from the minus end, which binds to structures composed of γ-tubulin.
80
Explain MT growth and dynamic instability.
Only ß-tubulin hydrolyzes its bound GTP, and this occurs when the tubulin dimer binds to an MT. Under conditions where there are plentiful free dimers, this hydrolysis occurs only after addition of further tubulin to the plus end of a growing MT. GDP-bound tubulin binds to the growing MT more weakly, but does not destabilize it if additional dimers have bound. Under conditions of limiting tubulin dimers, the GDP bound form is at the cap, and this has a destabilizing effect.
81
What do Colchicine/vinblastine do?
They are drugs that prevent MT polymerization.
82
What does taxol do?
It's a chemotherapeutics drug that inhibits MT depolymerization.
83
What do MT's do?
Microtubules form the mitotic spindle for chromosome separation during mitosis.
Microtubules are important for transport of organelles in all cell types – including long axons. Transport occurs using motor proteins which move their cargo towards the plus end (known as kinesins) or the minus end (known as dyneins)
84
What are cilia?
MT projections covered by plasma membrane. Used for movement of materials over cells.
85
What are flagella?
A single projection used to propel an entire cell with a beating motion.
86
What is the purpose of actin?
Actin is important for movement – of cells by crawling and in muscle contraction. Force
generation occurs by actin polymerization and also in conjunction with myosin motor proteins.
87
What is the structure of actin?
Actin forms filaments that have polarity (plus end, also called barbed end), and have more flexibility than microtubules. Actin filaments are often found in bundles or networks. The arrangement of actin filaments is regulated by a large number of actin binding proteins.
88
How does actin move?
Actin binds and hydrolyses ATP, which is important for the treadmilling function, where actin filaments grow at the plus end and shrink at the minus end.
89
How does cell crawling work?
Actin is concentrated just underneath the plasma membrane at the cell cortex in a network/meshwork. This meshwork gives the plasma membrane shape and its dynamic character allows for movement.
Actin polymerization pushes the plasma membrane outward in the form of sheets called lamellipodia and spikes called filopodia. Once these attach to a solid surface the cell body is dragged forward due to actin/myosin contractile bundles.
90
How do muscles contract?
Muscle is powered by actin and the motor protein called myosin II. Myosin II forms filaments with protruding head domains that bind directly to actin and stimulate sliding. In the context of the muscle fibre or sarcomere, this sliding motion is translated into a contraction. Activation of myosin binding to actin filaments in muscle is stimulated by Ca2+ influx from the sarcoplasmic reticulum.
91
What is the Extracellular Matrix?
The ECM functions to hold tissues together, provide cushioning (cartilage) and strength (tendons) and can also act as a reservoir for growth factors. The ECM comprises proteins and carbohydrates such as proteoglycans. All ECM components are highly networked with each other and with receptors on the cell surface such as integrins. The composition of the ECM is variable depending on tissue. Epithelial cells sit on a thin layer of extracellular matrix called a basal lamina.
92
How do epithelial cells divide different body cavities and provide barriers between the gut and the blood and the blood brain barrier
This is achieved via a polarized cellular organization. The polarization allows for different functions, such as absorption of nutrients on one membrane and their secretion from another.
93
What are tight junctions?
(occluding junctions) - seal epithelial cells together in sheets. Prevents passage of small molecules between cells.
94
What type of junction join cells to each other and to ECM?
Anchoring junctions.
95
What are the two types of anchoring junctions and what are their purposes?
1. adherans junctions/ desmosomes **Adherins=actin and desmosomes=IF's Hold cells together and are formed by cadherins.
2. Focal adhesions and hemidesmosomes bind cells to ECM.
* *For focal adhesions, integrin proteins bind to the ECM and attach to actin inside the cell. For hemidesmosomes, intermediate filaments bind inside the cell.
96
What is a communicating junction?
Gap junction. Cell:cell communication. 2-4 nm. Gap is spanned by connexins which are
channel-forming proteins (channel called connexon). Gap junctions allow for electrical coupling between cells.
97
What do cadherins do for cell adhesion?
Cadherins function in cell adhesion via anchoring junctions including adherens junctions and desmosomes.
Cadherins are large glycoproteins that link by a homophilic (ie. the same cadherin) mechanism. Cadherins link to the actin cytoskeleton via adapter proteins called catenins (eg. ß-catenin and α-catenin). Disruption of the cytoskeletal interaction via the adapters leads to loss of adhesion.
98
What do integrins do?
Cell matrix receptors on cells.
-Differ from other receptor types because they are abundant on cell surface and bind their ligands with low affinity.
-They activate signaling pathways upon ligand binding
Comprise α and ß subunits held together noncovalently. Both are glycoproteins. Ligand binding requires Ca2+ or Mg2+. Several genes for each subunit. 8 types of beta subunit and 18 types of alpha subunit.
99
What are selections for?
Selectins are lectins (carbohydrate binding) that mediate Ca2+-dependent cell:cell adhesion in the bloodstream. L-selectin found in white blood cells while P-selectin found in platelets.
Each binds to a specific carbohydrate on another cell. During inflammation, endothelial cells express E-selectin that binds to the carbohydrate on white blood cells and platelets. Selectins collaborate with integrins. Selectins mediate weak binding first, followed by stronger integrin- dependent binding.
100
Describe the basal laminae.
```
Flexible mats (40 – 120 nm thick) of specialized matrix that underlies epithelial cell sheets and tubes. Also found around muscle cells, fat cells and Schwann cells. The basal lamina (BL) separates these cells from surrounding connective tissue and serves as a highly selective filter. BL influences cell polarity, metabolism, survival, proliferation, differentiation and serve as highways for migration. BL is synthesized by cells that rest on it. In multilayered epithelia, such as stratified squamous epithelium of epidermis of skin, the BL is tethered to the underlying connective tissue via type VII collagen fibrils in a structure
that is called the basement membrane.
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101
What is responsible for cell weak adhesion and rolling?
Selectins
102
What is responsible for strong adhesion and emigration?
Integrin
103
How are nucleotides linked?
Nucleotides in a nucleic acid chain are linked from a 3’ hydroxyl to a 5’ phosphate, in a phosphodiester bond.
104
What are the 4 steps in gene expression at which regulation can occur?
transcriptional, post-transcriptional, translational, and post- translational.
105
DNA that is tightly wound around histones and is inaccessible to transcription machinery is referred to as?
Heterochromatin
106
Accessible DNA is referred to as?
Euchromatin
107
What helps the process of DNA packaging?
Polycationic amines. One example is spermine.
108
What replicates DNA prior to cell division?
DNA polymerase
109
What transcribes DNA into RNA?
RNA polymerase. (II for protein coding regions)
110
How does RNA Polymerase work?
RNA polymerase, which incorporates incoming ribonucleoside triphosphates (ATP, CTP, GTP, UTP) into a growing RNA chain. Only one strand serves as template, which is called the “template strand.” The complementary strand is called the “non-template” strand. Again, template is read in 3’-to-5’ direction; synthesis is in 5’-to-3’ direction.
Since both the RNA transcription product and the DNA non-template strand are complementary to the DNA template strand, the DNA non-template strand and the RNA have the same sequence (except that DNA contains T where RNA contains U). This is why, the DNA non-template strand is also called the “coding strand.”
111
Where does RNA Polymerase start?
The “promoter” (designated with a P and overhead arrow in the diagram below) is a DNA sequence that is recognized by RNA polymerase for binding and beginning transcription. More on promoters and related elements in the next lecture.
The base-pair at which initiation of txn takes place is defined as the “+1” site, or “transcription start site” (TSS). RNA polymerase synthesizes a transcription product, termed a “pre-mRNA,” which terminates well downstream at a “terminator” site. The entire stretch of DNA that is transcribed to give a pre-mRNA is referred to as a “transcription unit.”
112
What does splicing do to the pre-mRNA?
Splicing of pre-mRNA results in loss of introns (intervening sequences) and retention of exons (expressed sequences).
113
What are the elements of an mRNA sequence?
There is the coding region, which contains the nucleotide triplet codons that the ribosome will read to make a polypeptide chain. There are also two untranslated regions or UTR's at either end of the coding sequence deemed 5' and 3' UTR's.
114
How does txn initiation differ in RNA than in DNA?
Unlike DNA replication, where synthesis of new DNA strands proceeds from a pre-existing 3’ hydroxyl, RNA synthesis can begin de novo. (Details of the nucleic acid synthesis reaction will be shown in lecture.) Thus, the first nucleotide in the pre-mRNA is a nucleoside 5’-triphosphate. As we will see, mRNAs have a 5’ cap that is added to this 5’-triphosphate end.
115
How is RNA Pol activated?
Phosphorylation. Massive phosphorylation on its largest subunit site signals a switch to elongation mode.
116
What happens when the RNA Pol II reaches the polyadenlyation signal?
In response to the polyadenylation signal (see post-transcriptional processing lecture), a number of protein factors rearrange the elongating txn complex such that RNA pol II is released from the DNA template and is returned to its hypophosphorylated state by a CTD phosphatase. This is important to recycle RNA pol II for repeated rounds of txn.
117
What are the molecules that make up chromatin?
The major histone proteins form octamer histone cores, consisting of two each of H2A, H2B, H3, and H4.~ 150 base-pairs (bp) of DNA are wound around each histone core. An additional histone monomer, H1, binds to constrain the DNA to the core.
118
What is the direction of nucleic acid synthesis? In which direction is the template strand read?
Synthesis is 5'->3' and template strand is read 3'->5'
119
Name the types of sequences that are present in a pre-mRNA transcript.
Introns, exons, and UTRs.
120
What is a big difference between the way proteins enter the mitochondria and the nucleus?
Proteins enter the mitochondria in an unfolded state and the nucleus already folded.
121
Where do chaperones bind?
Hydrophobic regions of unfolded proteins.
122
How does the ER lumen environment compare to the cytosol?
The ER is an oxidizing environment. relative to the cytosol. Thus, sulfhydryl groups in the ER lumen are oxidized and disulfide bonds are formed. (S-S) In the cytosol, sulfhydryl groups remain in the reduced state. (-SH HS-)
123
Describe leukocyte migration into a wound?
Leukocyte migration into a wound begins with slowing of leukocytes by transient interactions between carbohydrate ligands on leukocytes and selectins on endothelial cells. Since selectins are anchored to actin filaments, any drug that interferes with actin function (choice a) will inhibit leukocyte migration. Subsequent to leukocyte slowing by transient selectin binding, stronger interactions occur with integrins expressed on endothelial cells. Thus, a drug that binds to an integrin (choice b) could interfere with leukocyte migration. After strong binding to endothelial cells, the leukocytes migrate between cells in the endothelial layer, and this requires cell movement via lamellipodia (choice e).
124
Where is the TATA box and what binds to it?
The TATA box, at which TFIID binds, is between -20 and -30.
125
What does the pre-mRNA sequence begin with?
Also note that the pre-mRNA begins with a 5’ nucleoside triphosphate (as indicated by ppp). Subsequent nucleotides are nucleoside monophosphates; the phosphate group is understood and not written in the sequence.
126
Describe the elements in the pre initiation complex (PIC)
Protein complexes called TFIIs (transcription factor for RNA polymerase II) bind DNA and each other to direct proper localization of RNA pol II for initiation of txn at +1. For example, upstream of the +1 site of some genes is a binding site for TBP (TATA-box binding protein), a subunit of TFIID. TBP binding resulting in a bending of the DNA, which is the first step in txn initiation. The TFIID complex consists of TBP and many TAFs (TBP-assoacited factors) In addition to the various TFIIs (A, B, D, E, F, H), another large protein complex called “Mediator” is also involved in txn initiation. The complete ensemble of protein complexes at the txn initiation site is called the “pre-initiation complex” (PIC).
127
Where does hyperphosphorylation of RNA Pol II occur?
The carboxy-terminal domain (CTD) of the largest RNA Pol II subunit.
128
What is a cis element?
In addition to the core promoter, a number of DNA “boxes” function as binding sites for proteins that will work to increase (or less commonly decrease) the rate of txn initiation. Consensus sequences on the DNA itself are called “cis elements” whereas proteins that bind such sequences or each other are called “trans elements.” cis elements that are bound by “constitutive” (= unregulated) transcription factors are generally located close to the core promoter and are called “promoter-proximal elements.” cis elements that are bound by regulated transcription factors are called “enhancers,” and these elements can be located quite far from the promoter
129
What are trans elements?
Txn factors. (more correctly called txn activator proteins) bind to specific target DNA sequences. Their presence on the DNA serves to enhance the rate of txn initiation by recruiting other proteins (called “co-activators”) that affect formation of the pre-initiation complex or accessibility of the DNA for txn initiation. There are at least two thousand txn factors encoded in the human genome.
130
What does a txn factor consist of?
A txn factor consists of protein domains, including a DNA-binding domain that recognizes a specific DNA sequence (often direct or inverted repeats), an activation domain that interacts with other proteins to enhance the txn initiation rate, and, often, a dimerization domain that allows formation of homo- or hetero-dimers (or tetramers) of txn factors.
131
How is there such variety in the ways txn factors can effect txnl regulation?
The human genome contains over 2,000 transcription factor genes, providing a level of complexity for transcriptional regulation that is expected for a complex organism. To increase further the possible combinations of transcription factors, these factors can bind as homodimers (or tetramers) or heterodimer (or tetramers). Thus, the actual number of transcription factor complexes available to bind to enhancers and influence the rate of transcription is even greater than the total number of transcription factor proteins.
132
Where can the txn factor activation domain bind to?
The txn factor activation domain can interact directly with constituents of the pre-initiation complex to increase the rate of txn initiation. In addition, the transcription factor activation domain can interact with co- activators that change the state of local chromatin – e.g., a histone modifying enzyme such as HAT (histone acetyl transferase), which acetylates histone lysine residue (activates it), which recruits other proteins that have domains that recognize acetylated histone tails and serve to attract basal transcription factors or chromatin remodeling complexes. Acetylation of histones is reversible, by the action of an HDAC (histone deacetylase) enzyme, which usually has an inhibitory effect.
133
what can chromatin remodeling complexes do in addition to chromatin modification?
In addition to chromatin modification, chromatin remodelling complexes can change the histone protein composition of the nucleosome or reposition nucleosomes on the DNA to expose regulatory sequences (as shown below). Multiple txn factors are typically involved in the regulation of a single gene.
134
what does dan methylation do?
Methylation of cytosine in a CpG dinucleotide generally acts to repress transcription. The mechanism is by recruitment of HDAC activity, by something like MeCP2
135
What are the main txn factors for B globulin transcription?
KLF1 and GATA1.
KLF1 is modified post-transcriptionally by phosphorylation at T41, and is inactive without this phosphorylation. An essential binding site for KLF1, known as a CACCC box, is found at -90 of the β-globin promoter, and multiple KLF1 binding sites are in an enhancer sequence located ~100 kb upstream of the β globin gene, known as the LCR or locus control region. KLF1 interacts with CBP, a large complex that includes a protein with HAT activity. It is thought that histone acetylation opens up the chromatin structure in the vicinity of the β globin gene promoter, allowing txn to increase >1000-fold. Expression of GATA1 is turned on at the same time as KLF1, and it also interacts with CBP. **Both GATA-1 and KLF1 are necessary for β-globin gene induction and neither is sufficient. GATA1 binds DNA as a monomer at various sites, including upstream of the β-globin promoter and in the LCR.
136
Mutations in the γ glob in regulatory region were shown to be associated with HPFH which suggest what?
That the mutation disrupted a repressor binding site.
137
Mutation of what gene results in ongoing transcription of γ glob in, even at late stages in erythroid development.
BCL11A
138
What regulates BCL11A
KLF1
139
What does the cap of an mRNA consist of?
The “cap” at the 5’ end of an mRNA consists of a guanosine nucleotide that has been modified to contain a methyl group at the 7 position of the purine ring structure. This is linked to the 5’ end of the transcript in an unusual 5’- to-5’ phosphodiester bond.
Immediately after RNA synthesis begins, the 5’ end of an RNA transcribed by RNA pol II is capped. The cap structure consists of a GTP molecule attached in an unusual 5’-to-5’ linkage to the 5’-terminal nucleotide of the pre- mRNA. The purine ring of this GTP is then methylated at the 7 position, giving 7-methylguanosine (abbreviated m7G). The first and second 2’-OH groups are also often methylated. The cap structure is required for translation initiation.
140
What is the purpose of the cap?
Only mRNAs (and not other cellular RNAs) are capped, and the cap serves as a recognition site for binding of proteins that recruit a ribosome. In addition, the cap functions to protect pre-mRNA and mRNA from degradation by exonucleases that chew in the 5’-to-3’ direction. Uncapping is one of the first steps in mRNA decay, which is now being appreciated as an important step in regulation of gene expression.
141
What is the polyA signal?
At end of the 3’ UTR sequence, a polyadenylation signal (most often AAUAAA) signals for endonucleolytic cleavage 10-30 nts downstream. The free 3’-hydroxyl is acted upon by poly(A) polymerase, a specialized RNA polymerase that adds 100-200 A residues in a template-independent fashion.
142
What is the poly(A) binding protein?
The poly(A) tail is bound by PABP, poly(A) binding protein. This protects the 3’ end from rapid degradation by 3’-to-5’ exonucleases and, as we will see, enhances translation via an interaction between the 3’ end and the 5’ end of the message.
143
What are the conserved sequences of introns?
Almost universally, introns start with a GU dinucleotide and end with an AG dinucleotide. These dinucleotides are recognized in the context of loose consensus sequences that include exon and intron sequences.
Introns contain a “branch point” near the 3’ splice site, which is an A residue in the context of a loose consensus sequence, as well as a run of pyrimidines also near the 3’ splice site.
144
What catalyzes splicing?
A spliceosome, a large complex of nearly 200 proteins and 5 snRNPs.
145
How does splicing occur?
The 2-OH from the A branch attaches the 5' splice site in a transesterification and then the 3-OH of that 5' piece attacks the 3'splice site essentially cutting out the intron.
146
What do SR proteins do?
They bind at exotic splicing enhancer sequences which are in proximity to the correct 5’ GU and 3’ AG dinucleotides of the splice junctions and SR binding is how the splicing machinery recognizes it's in the right place.
147
Why is the human trasncriptosome so much more complex than the human genome?
The current estimate for the number of genes in the human genome is ~25,000. That is not a lot of complexity for such a complex organism. However, each gene transcript can undergo alternative splicing to give multiple mRNA products. It is thought that transcripts from >90% of genes undergo alternative splicing. Thus, the human “transcriptome” is vastly more complex than the human genome.
148
What are the types of alternative splicing?
The most common form of alternative splicing is exon skipping. Other forms include alternative 5’ splice site, alternative 3’ splice site, and intron inclusion.
149
how is alternative splicing regulated?
Alternative splicing is regulated by SR proteins, whose own activity is regulated by several mechanisms, such as phosphorylation, localization, and level of synthesis. In addition to ESEs, exons may contain ESSs, which are exonic splicing silencers. These are RNA boxes that bind SR proteins to suppress splicing of an exon, such that it is not included in the final mRNA product.
150
What are ori?
he large size of the human genome, and the need to complete replication within a limited time in the cell cycle, mandates that each chromosome has multiple sites at which DNA replication initiates. These sites are origins of replication (called “ori”), and they are located on average 100,000 base pairs (bp) apart.
151
Which direction does DNA synthesize from an ori?
DNA synthesis starts at an ori and the “replication fork” representing newly synthesized DNA moves away from the ori in both directions simultaneously.
152
How does bidirectional replication happen?
All nucleic synthesis occurs only in the 5’-to-3’ direction. Bi-directional DNA replication away from an origin is therefore semi-discontinuous, i.e., on one template strand DNA polymerase synthesizes new DNA continuously (the “leading” strand), and on the other template strand DNA polymerase synthesizes new DNA in short Okazaki fragments (the “lagging” strand).
153
What does DNA primes do?
Unlike synthesis by RNA polymerases, DNA synthesis proceeds only from a pre-existing “primer” that provides a free 3’ hydroxyl onto which DNA polymerase adds the next nucleotide. DNA primase, a subunit of DNA pol α (see fig. below) is an RNA polymerase that lays down a short RNA primer, which is then extended by DNA polymerase.
154
What cleaves off the RNA primer during DNA replication?
FEN1, a flap endonuclease. The nick between the 5’ end of the “old” Okazaki DNA and the 3’ end of the “new” Okazaki DNA is sealed by DNA ligase.
155
What are the different kinds of DNA polymerases and what do they do?
DNA pol ε, which synthesizes the leading strand, and DNA pol δ, which synthesize the lagging strand, according to recent models. Synthesis of the RNA primer and primer extension to start replication are accomplished by DNA pol α. Smaller DNA polymerases, such as DNA pol β and others, are involved in DNA repair, which will be discussed in the next lecture. Mitochondrial DNA is replicated by DNA pol γ.
156
Describe a telomere.
Telomeric DNA is consists of a long stretch of DNA (several thousand base pairs) that have the repeating sequence TTAGGG. This DNA folds back into a structure called a “T-loop” (see figure below)that distinguishes it from a broken DNA end (which is a signal of catastrophic DNA damage that can result in cell suicide or apoptosis). The T-loop also prevents end-to-end joining of chromosomes.
Telomere repeat sequences are bound by telomere-specific proteins that form the “shelterin” complex.
- The extended G-rich strand folds back and anneals to the C-rich strand, creating a local displacement loop (or D-loop), resulting in the overall T-loop formation.
- Binding of repeated shelterin complexes protects the telomere end.
157
What is the end replication problem?
The nature of lagging strand replication means that the very end of the lagging strand cannot be fully synthesized, as shown below. Without a mechanism to repair this problem, telomeres would shorten at each round of replication, resulting in an inability to form the T-loop and inappropriate DNA repair as well as end-to- end joining.
158
What solves the end replication problem and how?
To solve the end replication problem, cells use a reverse transcriptase known as “telomerase,” a ribonucleoprotein complex that carries its own RNA template and that can extend the lagging strand. Normal, differentiated somatic cells do not have telomerase activity, and they therefore can undergo only a limited number of cell divisions before they become quiescent. This is known as the “Hayflick limit.” In some cancers, the reactivation of telomerase allows cells to continue to divide unchecked.
159
What is the RNA polymerase that lays down a short RNA primer, which is then extended by DNA polymerase
DNA Primase
160
Why would an inhibitor of viral DNA polymerase not also inhibit cellular DNA polymerase, since both
catalyze the same DNA synthesis reaction?
For example, the HIV life cycle includes reverse transcription of genomic RNA into double-stranded DNA that then integrates into the chromosome. Nucleoside analogues that do not have a 3’ hydroxyl onto which a subsequent nucleotide can be added are used as antivirals.
161
How are modified bases most often repaired?
Repair of modified bases most often occurs by a system called “base excision repair.” Glycosylases that recognize unnatural bases in DNA (e.g., uracil glycosylase) patrol the genome, cutting the bond between the base and the 1’C of the ribose sugar to create an abasic site. This is recognized by other enzymes, the abasic nucleotide is removed and the correct nucleotide is filled in
162
What repairs methylated bases?
Methylated bases (e.g., O6-methyl guanine can pair with T instead of C) can be repaired by a “direct reversal” protein, which binds to the methylated base and transfers the methyl group to a Cys residue in its active site.
163
What kind of DNA lesions can UV light cause?
Pyrimidine dimers, where consecutive pyrimidine rings are covalently linked to each other. This causes a terrible kink in the DNA, blocking replication and transcription.
164
How are pyrimidine dimers fixed?
The “nucleotide excision repair” system deals with pyrimidine dimers. An XP protein recognizes the distorted DNA region, other XP proteins unwind and excise a patch of DNA, which is then filled in by DNA pol ε or δ.
Alternatively the pyrimidine dimer is dealt with by a process called “translesion synthesis.” Different DNA polymerases (e.g., η, ι), which are not processive and are error-prone, are able to replicate past the pyrimidine dimer, either putting in the correct bases opposite the dimer or incorrect ones. The disadvantage of insertion of an incorrect base is far outweighed by the disastrous effect of a block to replication that would occur in the absence of repair and translesion synthesis.
165
What type of damage does X-ray radiation confer to DNA?
Exposure to X-rays can result in double-stranded DNA breaks. A break in a chromosome has important consequences, since this is incompatible with the cell’s mechanism of equally distributing chromosomes to daughter cells. This is trickier to repair because there's no template to base repairs off of.
166
How are double stranded breaks repaired?
Such damage is usually repaired by non-homologous end-joining (NHEJ), in a process that involves imprecise ligation of the broken ends. A less common form of repair is via homologous recombination, using the other chromosome copy as the basis for repair of the broken one.
167
What is unequal crossing over?
Two or more copies of the same gene can be present on a chromosome. If homologous copies of the gene align out of register and undergo recombination, the result will be unequal distribution of gene copies, which may cause disease.
168
What is transposition?
Transposition is the movement of a unit of DNA called a “transposon” from one site to a new target site. Unlike homologous recombination, transposition occurs in the absence of homology, and the insertion generally occurs at random sites. It is catalyzed by a transposase enzyme, which is often encoded by a gene located on the transposon itself. As uncontrolled transposition would wreak havoc with genome structure, this process is highly controlled via regulation of transposase gene expression.
169
How does bacterial transposition occur?
We will mention only one of two mechanisms for movement of bacterial transposons, the cut and paste (non- replicative) mechanism. The ends of the transposon unit in the donor DNA are recognized by transposase, which makes cuts at those ends. The transposase also makes staggered cuts in a target DNA site. The transposon DNA is inserted in the target site, which could be on the same DNA molecule as the donor or on a different DNA molecule.
170
How do bacterial transposons contribute to antibiotic resistance?
A bacterial transposon can be a simple “insertion sequence,” carrying nothing but the transposase coding sequence (see example of IS3, below). It can also be a complex transposon, which carries, in addition to the transposase coding sequence, a gene for antibiotic resistance, for example. See below for two examples of bacterial transposons carrying an ampicillin resistance gene (Tn3) or a tetracycline resistance gene (Tn10). The facile spread of these elements accounts for multiple drug-resistant strains of bacteria that are a large problem in hospitals.
171
What is the difference between bacterial transposons and eukaryotic transposons?
While bacterial transposons move via a DNA-only mechanism, eukaryotic retrotransposons move via an RNA intermediate. The transposon DNA sequence is transcribed by RNA polymerase, creating an RNA copy of the transposon sequence. This is followed by reverse transcriptase conversion of the RNA intermediate to double- stranded DNA, which then integrates into the target DNA via the activity of an integrase protein. The retrotransposon sequence includes the coding sequence for reverse transcriptase.
172
What are LINES?
A non LTR (long term repeat) DNA transposon. LINES (long interspersed elements)
The human genome contains about 500,000 copies of an element termed “L1.” The full-length element is about 6,000 bp long, but the vast majority of L1 elements are truncated. Even of the full-length elements, most are mutated to the point of non-functionality, and there are only about 50 active L1 elements. These code for a protein that has reverse transcriptase activity. Retrotransposition by an L1 element is known to be the cause of disease in several cases. More importantly, an L1 element provides a region of homology at which recombination can take place.
173
What is the significance of SINES?
The major type of SINE in the human genome is the AluI element, a 300-bp DNA that is present in about a million copies. These contain no coding sequence, and their transposition depends on the assistance of L1 elements. Here, too, movement of an AluI element can cause disruption of a gene and disease.
174
What is the structure of tRNA?
The link between mRNA and protein is a small nucleic acid that has a 3-nt complement to a codon in the middle of the molecule (the “anticodon”) and an amino-acid residue covalently linked at the 3’ end.
175
What is used for translation initiation?
A special form of methionyl tRNA is used for translation initiation.
176
What attaches an amino acid to its cognate tRNA (the process of tRNA “charging”) ?
aminoacyl tRNA synthetase
177
tRNA charging is accomplished at the expense of what?
Two high energy phosphate bonds. (ATP-> AMP)
178
What are the steps in tRNA binding?
aminoacyl tRNA synthetase facilitates the bonding of the appropriate amino acid to it's tRNA using the energy from 2 phosphate bonds. The tRNA can then bond to the appropriate codon on the RNA.
179
What performs translation?
Translation is performed by a ribosome consisting of two subunits (60S, 40S). that each contain dozens of proteins and a large quantity of rRNA (hence the name “ribosome,” for ribonucleic acid). Ribosomal RNA functions as the actual enzyme of peptide bond formation, with the proteins playing a structural role.
180
What are the 3 sites on the ribosome and what is their function?
The A site (aminoacyl site) is where the next charged tRNA binds. The P site (peptidyl site) is where the tRNA with the growing peptide chain is located. The E site (exit site) is where the tRNA in the P site goes after its peptide cargo has engaged in peptide bond synthesis.
181
What is required to bind the ribosome to the mRNA 5’ end and position the ribosome for correct initiation of translation.
Eukaryotic initiation factors. (eIFs)
182
What is an eEF and what does it do?
eEFs (elongation factors) participate in bringing succeeding charged tRNAs to the site on the ribosome where peptide bond formation takes place, as well as translocation of the ribosome to the next codon.
183
What is required for the release of the finished polypeptide upon encountering a stop codon, and for release of the ribosome from the mRNA?
A single eukaryotic releasing factor (eRF).
184
How is translation initiated?
Interaction of eIFs with 5’ cap forms a recognition site for ribosome binding. The ribosome “scans” in the 5’-to-3’ direction for an AUG that is in the correct sequence context to function as a start codon. The start of the codon sequence defines the end of the 5’ UTR. In association with a number of eIFs (eukaryotic initiation factors) and the special initiator fMet-tRNA, the small 40S ribosomal subunit recognizes the eIF4-PABP complex bound at the 5’ cap of an mRNA. At the expense of ATP hydrolysis, the 40S subunit scans downstream until it finds an AUG start codon in the correct context (Kozak model), forming the pre-initiation complex.
185
Draw translation initiation.
See 10th screenshot.
186
What is dwell time and why is it important?
Incorporation of the “correct” amino acid in the growing peptide chain depends on the dwell time of a charged tRNA with the codon. A longer dwell time, due to correct complementarity of the anticodon and codon, allows the GTP-bound eEF1A, which brings the tRNA to the A site, to hydrolyze GTP. GTP hydrolysis is required for eEF1A to leave (as the GDP-bound form), allowing peptide-bond formation.
187
What is the energy investment to incorporate 1 amino acid into a peptide chain?
It requires the hydrolysis of 4 high-energy phosphate bonds.
In addition to the hydrolysis of GTP required to release eEF1A and allow peptide-bond formation, the movement of the ribosome down one codon requires hydrolysis of a GTP in the translocase reaction. Thus, incorporation of one amino acid in a growing polypeptide chain requires hydrolysis of four high-energy bonds: two in the aa-tRNA synthetase reaction and two in the elongation reaction.
188
What leads to the 3', 5' interaction in mRNAs and what is the point?
A ribosome arriving at a stop codon is released from the mRNA. The 3’ end and 5’ end of an mRNA are connected via the interaction of poly(A)+ binding protein and eIFs that bind the 5’ cap, which allows ribosomes that have been released from the mRNA to rapidly bind again at the 5’ end to re-initiate translation. “Polysomes” are formed when multiple ribosomes are translating a single mRNA, and active translation of a particular mRNA is revealed by its partitioning into the “polysome fraction.”
189
Why is it important that there are differences in human and bacterial ribosomes?
Although the structure and function of ribosomes is highly conserved throughout life, there are differences between the bacterial and human ribosomes that enable the use of antibiotics that interfere with bacterial translation but not with human cellular translation.
190
How do cells under stress regulate protein translation?
Inhibition in response to stress.
Cells respond to many forms of stress by activation of one of a number of protein kinases that phosphorylate eIF2, an initiation factor that is required to present initiator tRNA to the ribosomal P site. Phosphorylated eIF2 cannot switch from the inactive GDP-bound state to the active GTP-bound state.
Some of these are PERK/PEK, PKR, GCN2, and HRI/PfPK4.
191
How can eIF's respond to growth signal?
The activity of eIFs is regulated by factors that bind to them. For example, eIF4E, is part of the eIF4 complex that binds the 5’ cap and is required for ribosomes to recognize mRNA for translation initiation. In the baseline state, several 4E-BPs, or eIF4 binding proteins, down-regulate eIF4 activity. However, when the cells receive a growth factor signal and enter a cell division mode, which requires a high level of protein synthesis, the mTOR complex phosphorylates 4E-BPs, releasing them from the eIF4E complex and allowing vastly increased translation.
192
Describe how the translation of ferratin can be regulated.
Regulatory proteins may bind to sites in the 5’ UTR and interfere with ribosome scanning. Such sites can be hairpin stem-loop structures that form by intramolecular base pairing. The example shown below involves expression of ferritin, an iron storage protein. When there is abundant iron, a protein named IRE-BP (iron regulatory element binding protein) does not bind the 5’ UTR of ferritin mRNA and translation proceeds. When iron is scarce, and there is no need for ferritin, IRE-BP binds the ferritin mRNA 5’ UTR and translation is blocked.
193
How can regulatory proteins target mRNAs for degradation?
Regulatory proteins may also bind to specific features in the 3’ UTR, recruiting an endonuclease that cleaves
the message and exposes a 3’ end for rapid degradation by 3’ exonucleases.
194
How do micro RNA's work?
Small (~20nts) double-stranded RNAs, known as microRNAs, are produced in the cell from larger precursors. A mature microRNA is introduced into the RISC complex (RNA-induced silencing complex), where the double-stranded RNA is unwound and the single-stranded RNA guides the RISC complex to a specific target mRNA. The target mRNA is either cleaved or its translation is inhibited.
195
Describe the process of miRNA production and function. Include helpers.
The canonical pathway for miRNA production in humans. Step 1: An RNA polymerase II enzyme is responsible for reverse transcription of the gene, and the resulting molecule is a single-stranded RNA with a stem-loop secondary structure called the primary miRNA (pri-miRNA). Step 2: The pri-miRNA is processed within the nucleus by RNASEN and DGCR8, which combine to form the microprocessor complex. Step 3: The resulting RNA molecule is termed preliminary miRNA (pre-miRNA). Step 4: Exportin-5 binds pre- miRNA and shuttles the molecules through nuclear pores to the cytoplasm, a process that requires Ras- related nuclear protein (RAN), a guanosine triphosphatase. Step 5: In cooperation with its cofactor
TRBP, DICER1 functions in a manner analogous to the nuclear microprocessing complex by measuring the necessary length of the miRNA and cutting the pre-miRNA to mature 20- to 22- nt strands. Step 6: The resulting mature miRNA is then passed on to a member of the Argonaute family and other participating proteins, including Gemin3, Gemin4, and GW-182. Step 7: Argonaute binds the miRNA and forms RISC, a group of proteins that is directed to the mRNA transcript to be silenced and either cuts the transcript to prevent translation or recruits other proteins to block translation. DRAW THIS OR LOOK AT screenshot 11 to help remember.
196
Why is the discovery of iRNA or miRNA so powerful?
since introduction of exogenous siRNA (small interfering RNA) allows “knock- down” of expression of a target gene. Targeted knock-down of gene expression is viewed also as a potential pharmaceutical to treat disease. Problems with siRNA delivery, lifetime in vivo, and specificity (“off-target effects” may occur) are still troubling the field.
197
Explain how the ribosome is a ribozyme.
The 1982 discovery of ribozymes demonstrated that RNA can be both genetic material (like DNA) and a biological catalyst (like protein enzymes), and contributed to the RNA world hypothesis, which suggests that RNA may have been important in the evolution of prebiotic self-replicating systems. Also termed catalytic RNA, ribozymes function within the ribosome (as part of the large subunit ribosomal RNA) to link amino acids during protein synthesis, and in a variety of RNA processing reactions, including RNA splicing, viral replication, and transfer RNA biosynthesis. Investigators studying the origin of life have produced ribozymes in the laboratory that are capable of catalyzing their own synthesis under very specific conditions, such as an RNA polymerase ribozyme.
198
List the enzymatic steps of DNA base excision repair.
DNA glycosylase: takes bad base out
AP endonuclease: identifies and cuts the backbone
DNA Phosphodiesterase: widens the gap to make room for new, correct base pairing.
DNA polymerase/ligase: correct insertion of base and connection to the rest of the chain.
199
What can repair with DNA base excision from oxidative stress?
8-oxoguanine DNA glycosylase (OGG1)
200
How does UV damage affect DNA
It creates nucleotide dimers.
201
How are nucleotide dimers fixed?
Nucleotide excision repair. Nuclease exists the dimer with XP gene products. DNA pol E fixes and ligase ties.
202
Draw out process of translation initiation.
Best diagram is screenshot 12.
203
How does molecular mimicry help in translation termination?
Basically eRFs serve as a molecular mimic to tRNAs and have a similar stop codon domain that allows them to behave like a tRNA and terminate translation.
204
What does a transcription factor actually do?
Transcription factor binding serves to increase the rate at which RNA polymerase initiates transcription, i.e., how often per unit time transcription begins. Transcription factors do not affect the rate of RNA polymerase transit down the DNA template.
205
```
What are the functions of these:
DNA methyltransferase
B. histone acetyltransferase
C. DNA helicase
D. TFIIH kinase
E. DNA topoisomerase
```
DNA methyltransferases are enzymes that methylate DNA at the C residue of a CG dinucleotide. Such methylation is associated with a decrease in gene expression, due to the binding of MeCP2 to methylated DNA, which recruits a complex containing HDAC (histone deacetylase) activity. The latter activity, removal of acetyl groups from the lysine residues of histone proteins, is associated with decreased gene activity. Choices b-e are activities associated with active gene expression: Modification of histones by HAT (histone acetyltransferase) activity results in a weaker histone-DNA association and more accessibility to transcription machinery. DNA helicase and DNA topoisomerase are needed to unwind DNA ahead of transcribing RNA pol II. Phosphorylation of RNA Pol II by the TFIIH kinase is required for initiation of transcription.
206
How do mutations at or downstream of the +1 start sight affect transcription?
Mutations in sites that are at or downstream of the +1 transcriptional start site (all other choices) would not be likely to affect transcription.
207
Where do shorter mRNAs fall on a northern blot?
Lower. Shorter sequences travel faster.
208
What is important to keep in mind when looking at the molecular weights on PCR blots?
The length of the primers too.
