Gene regulation Flashcards
(32 cards)
- Explain the natural structures that DNA forms in response to tension
- DNA in the cell is usually under tension (over or underwound), forming supercoils
- This is regulated by the cell
- Most cellular DNA is underwound
- (Overwound would be when there are more turns for the same amount of DNA)
- The natural tendency of DNA that is under strain is producing another curve - supercoil
- Supercoiling forms when it is under tension to actually relieve the tension
- Interestingly, when DNA is underwound its easier to seperate the strands
- e.g. if you seperate the threads, that puts tension further along the strand that can only be relieved by forming supercoils
- so whenever DNA is being seperated (e.g. transcription), you get areas of overwound and underwound DNA - these are under tension and the tension must be relieved
- Positive supercoiling = DNA overwound - occurs ahead of replication fork
- Negative supercoiling = DNA underwound - easier to seperate strands
Note: pos and neg dont refer to DNA charge - Topoisomerases relieve this tension
- Explain why a circular DNA forms a ladder when run on an agarose gel
Supercoiling can be observed on an agarose gel
- gel electrophoresis seperates DNA on the basis of size (only for linear DNA though)
- For circular DNA, both its size and shape are considered
1. relaxed circular DNA takes a long time to travel through the gel (larger volume)
2. Supercoiled circular DNA will travel through the gel much faster (act like linear DNA) (more compact - less resistant)
AFTER TREATMENT OF DNA WITH TOPOISOMERASES:
- makes bands (ladders) that represent different number of supercoils
- Each band are identical pieces of DNA but instead of being different sizes, they are different shapes because of different n.o supercoils
- Describe the role of DNA topoisomerases in the cell
MECHANISM OF TYPE I TOPOISOMERASE:
🧬 Mechanism of Type I Topoisomerase:
Type I Topoisomerases relieve supercoiling in DNA by making a transient single-strand break in the double helix.
🔧 Step-by-step:
The enzyme binds to double-stranded DNA and separates the strands slightly.
It uses a tyrosine residue in its active site to cleave one strand of the DNA:
The OH group on the tyrosine attacks the phosphodiester bond in the DNA backbone.
This forms a covalent bond between the tyrosine and the phosphate of the DNA → this protects the DNA end from degradation.
The unbroken DNA strand passes through the break.
This relieves torsional strain (supercoils).
The cleaved DNA strand is resealed:
The free 3′ OH of the broken DNA attacks the phosphate linked to the tyrosine.
This re-forms the phosphodiester bond, releasing the tyrosine.
🧠 Key Points:
No energy (ATP) is needed for Type I topoisomerase.
The reaction is reversible and precise, leaving no mutation or loss in the DNA sequence.
- this results in a bond between tyrosine (amino acid) and a base (nucleic acid) –> covalent linkage between protein and DNA
- Explain the mechanism of a Type II Topoisomerase
🧬 Mechanism of Type II Topoisomerase (e.g., DNA Gyrase):
Function: Relieves positive supercoils or introduces negative supercoils by cutting both strands of DNA and passing another double-stranded segment through the break.
Requires ATP (unlike Type I).
🔧 Step-by-Step Mechanism:
Binding:
The enzyme binds a segment of double-stranded DNA → this is the G segment (Gate segment).
The G segment is held at the C-gate (bottom part of the enzyme).
Capture of T segment:
A second DNA segment (called the T segment, Transport segment) is captured at the N-gate (top part of the enzyme).
ATP binding causes the N-gate to close, trapping the T segment.
Double-Stranded Cut:
The enzyme uses two tyrosine residues to cleave both strands of the G segment.
The ends of the G segment remain covalently attached to the enzyme to protect the DNA.
Passage:
The T segment (held at the N-gate) is passed through the break in the G segment and exits through the C-gate.
Resealing:
The enzyme reseals the double-strand break in the G segment using energy from ATP hydrolysis.
Reset:
The enzyme resets to its original conformation, ready for the next cycle.
- Describe the structure of a nucleosome
DNA is protected within the cell by ALWAYS being bound to proteins
- Chromosome is 1/3rd DNA and 2/3rds protein by mass
> Important type of protein found in the chromosome is the HISTONE
2 loops of DNA are wrapped around a small core of histones:
- There are 8 histones per core = nucleosome
This is the first level of compaction of the chromosome - when DNA is loosest
DNA is nucleosomes is ~7 fold shorter
- Describe the role of histones in chromatin organisation
Histones are small BASIC proteins and have 5 classes:
highly conserved across species
- CORE of nucleosome is made of 8 histones:
> 2H2A, 2H2B, 2H3, 2H4 histones
*HISTONE 1 is on the outside of the nucleosome
- Histone 1 keeps the loop of DNA closed and stable to stabilise the whole nucleosome
- Explain the importance of the amino-terminal tail of histones for regulation of chromatin structure
Histone tails protrude from nucleosome
- these are amino tails that are flexible and disordered
- The tails poke out of nucleosome and allow 2 things:
1. Histone tails of neighbouring nucleosomes to interact with each other –> structure of chromosome is coordinated between different nucleosomes
2. Tails can be bound by enzymes that can modify them –> modification of these are important for regulation of chromatin
Histones are covalently modified:
- modifications occur on the histone tails and in the body of histones as well
- these are supposed to be part of a histone code which marks the DNA for specific biological processes
- These changes alter:
1. Structure and packing of chromatin
2. The access to the DNA of DNA binding proteins
Lysine acetylation:
- allow for the opening and closing of chromatin that then allows for DNA transcription
- Histone Acetyltransferases transfers acetyl groups on to histones and this allows nucleosomes to move apart and that whole area of chromatin becomes decondensed –> transcription can occur in these regions
- Define the roles of Histone Deacetylases (HDAC) and Histone Acetyl Transferases (HAT) enzymes in regulating chromatin structure and gene expression
Regulation of Histones for Chromatin Remodelling:
1. Chromatin remodelling complexes can carry this out on the chromatin, e.g.
- change sections of nucleosomes by spacing them out more
- remove sections of histones
- place histone variants (histone replacement) - e.g. alternate type of H3
- This will change the way chromatin acts
- Histone modifying enzyme modifies histones - e.g. Lysine acetylation (covalent modifications)
- these modifications will change the accessibility of that DNA to a whole series of enzymes that can carry out activities like transcription and replication
- these might bind to a particular region of chromatin that has this “code” and it will bring other proteins that can change what happens to the DNA so it can mark areas for silencing active gene or even as damaged to allow DNA repair enzymes to come and fix it
- Describe the levels of organisation of the DNA into a eukaryotic chromosome
Compaction/condenstion of chromosomes - levels:
1. First Level
- Histones + DNA = Nucleosome —> It doesnt get any looser than this in the cell!
- ~7-fold compaction
- Active DNA –> gene on a particular section of DNA can be transcribed
- Second Level
- Nucleosome + 1 Histone H1 wrapped into another coil –> not clear how it works
- 30 nm fibre
- ~100-fold compaction
- inaccessible DNA —> any gene in this section of DNA is silenced
- If we were to take a chromosome and loosen it chemical treatment we would see these 30nm fibres looping out of structure
- If we loosened it more with chemical treatment the DNA would spill out but we still see a structure of the chromosome - Protein scaffold (chromosomal scaffold) that controls the loops of 30nm fibres
- DNA loops come out of a protein scaffold
- each one of those loops is potentially being transcribed or not being transcribed –> they could be in different configurations
Areas of gene activity are not as tightly packed:
- This image shows the 30nm fibre (second level of compaction) and areas of gene expression are in their first level of compaction form –> transcribed –> active region of DNA
- Describe the changes to chromatin structure during the cell cycle and for cellular function
- Describe how DNA methylation changes chromatin structure and gene expression.
- EUKARYOTES:
- DNA itself can be modified
- So the base cytosine of DNA is methylated at its 5’ end to make 5-methylcytosine
- This has no effect on base pairing
- This methylation of cytosine occurs on CG dinucleotides when they occur on an extended region
*these occur in large regions with lots of CG repeats (linearly) - so they are all methylated –> called a CpG island - cytosine on opposite strand will also be methylated
- CpG are usually found in promoters and regulate transcription levels
- Although methylation of the cytosine in CpG islands doesn’t affect base pairing, it affects what proteins can bind to the DNA
- DNA methyltransferases are enzymes that can bind to those CpG islands to carry out the cytosine methylation to shut down the expression of the gene - the enzyme is directed to specific areas of the DNA by DNA specific binding proteins
> Methylation of the specific CpG islands is inherited by the Daughter cells - Daughter cell DNA will have the same methylation pattern as the Parental cell
(Not all CpG dinucleotides are methylated)
- When DNA is replicated, only one strand contains the methylation
- So maintainence DNA methyltransferases will recognise these hemi-methylated sequences and methylate the other strand
> These changes are called epigenetic:
- Doesnt affect DNA base pairing and sequence
- Changes are maintained and can be altered by signals
- Inherited cell to cell
- It also includes the acetylation of histone tails in the nucleosome
- Explain why tight regulation of gene expression is important for prokaryotes
- Genes are regulated to express certain genes for given environmental conditions
- The (-35) and (-10) regions of the promoter sequence just upstream of the coding region is important because it is where the RNA polymerase binds
EXTRA INFO:
[- RNA polymerase holoenzyme (core enzyme + sigma factor) binds to the -35 and -10 regions.
- Sigma factor helps the polymerase recognize and bind the promoter.
- After binding, the DNA unwinds at the -10 site to allow transcription to start at +1.]
- Additionally, there are other sequences in the DNA where other proteins can bind to control gene expression
- ACTIVATORS - activate transcription —> transcription factors
- REPRESSORS - repress transcription
- Bacterial genes are often controlled together in operons - one mRNA is produced and will contain three genes that are translated into 3 proteins
- Describe the basic process for the regulation of transcriptional initiation in prokaryotes
- Upstream of the promoter and regulatory region is a trp operon containing 5 genes: TrpE, tRPd, TRPC, TRPB, TRPA
- When tryptophan levels are low, the operon is transcribed and then translated to produce the 5 proteins that are enzymes important in the biosynthesis of tryptophan - the final product
The gene is expressed when there is no TRYPTOPHAN in the environment the E.coli grows in
> Another gene - TrpR is present uptream of the promoter region and it produces an inactive repressor
- if TRYPTOPHAN is present in the environment, it binds to the trpR region and activates it —> tRP is a corepressor that binds to a repressor protein and activates it so the repressor protein binds to the operator and inhibits transcription
- This active repressor can now bind to operator sequence of operon and block transcription
🔬 Structural Note:
- Tryptophan binding increases the distance between the DNA-binding helices in the TrpR dimer → this aligns them perfectly to fit into the major grooves of the operator DNA.
- This conformational change is what enables DNA binding.
🧠 Summary:
TrpR = gene → makes inactive repressor protein.
Tryptophan = corepressor → binds to TrpR protein → activates it.
Active TrpR binds to operator → blocks transcription.
It’s a negative feedback loop to avoid overproduction of tryptophan.
- Explain how transcription initiation of the trp operon is controlled
Above
- Predict how a mutation in different parts of the trp operon would affect gene expression
Yes - tute
- Explain how transcription initiation of the lac operon is controlled
- Lac operon encodes for an ACTIVE repressor by default unlike Trp operon which encodes for and INACTIVE repressor protein from its regulatory gene
- Tryptophan is a COREPRESSOR while Allolactose is a inducer - both occur by negative regulation however opposite ways
Negative regulation - when a molecule binds to a protein to decrease its activity (if its active - it’ll turn inactive)
- LacI is the regulatory gene that encodes for the active Lac repressor - the active repressor binds to the operon to inhibit transcription - this occurs in the absence of lactose
- In the presence of lactose, a form of lactose (allolactose) binds to the active repressor protein and inactivates it so transcription can occur
> Lac operon contains of 3 genes: LacZ, LacY, LacA
- These genes B-galactosidase, permease, transacetylase
WHAT DO THESE GENES DO?
1. B-galactosidase - breaks lactose into allolactose and glucose so E.coli can use it as an energy source
2. Permease - allows lactose to enter cytoplasm of E.coli
3. Transacetylase - DONT KNOW FUNCTION
- Predict how a mutation in different parts of the lac operon would affect gene expression
Yes - tute
- Explain the relationship between the quaternary structure of Lac repressor and the DNA sequence that it binds
What exactly is a transcription factor?
- Lac repressor is a transcription factor
- Protein that binds to DNA to regulate transcription
Mechanism of repression by the Lac repressor:
- The sequence bteween O3 and O2 is the operator sequence and surrounds the LacZ gene
- The promoter is between O3 and O1
[O3-O1-O2]
- All operator elements are palindrome sequences that read same backward and forward
- Lac repressor has a functional structure as a dimer
- Each monomer of that dimer binds to each palindrome element on either strand
- Transcription factors commonly bind like this to operator sequences - via palindrome as a dimer
- Lac repressor actually binds to 2 operator elements at same time - works as a tetramer with one dimer binding to each element (O3 & O1)
- When lac repressor binds to 2 operator elements it forms a loop in the DNA between it and prevents RNA polymerase from transcribing the lac operon
- Explain the combinatorial control of the lac operon in response to lactose and glucose
- Most genes are regulated by multiple transcription factors
- Many sites upstream of promoter where transcription factors can bind - either activators or repressors
- EXPRESSION OF GENE IS AFFECTED BY ALL OF THEM:
- Whichever transcription factors are bound to gene, together effect whether RNA polymerase binds and transcription occurs - all messages are integrated and interact to produce a single response (yes or no transcription)
> Gene activation is synergistic (interaction of two or more transcription factors to cause the effect):
- For example for a gene with two regulatory sites:
- if one transcription factor binds, it causes little bit of transcription
- if two binds, it increases transcription —> synergistic
- Predict the level of expression of the lac operon (and the binding of the transcription factors) under different growth conditions
- Lactose is a good energy source, but glucose is better
- Lactose takes more energy input to breakdown than glucose does
- If E.coli has both lactose and glucose, it is more efficient to use glucose before lactose —> that is what this regulation achieves
GENERAL:
- lactose repressor is inactive because of allolactose binding and hence in presence of lactose
- Simulaneously, cAMP levels (modification of ATP) are high because of low glucose levels (HIGH CAMP = LOW GLUCOSE)
- cAMP binds to inactive protein CAP (or CRP - same thing) and activates it
- Active CAP binds to promoter of lac operon
Note: CAP doesnt actually bind to operator like repressor proteins do, it binds to a section of promoter (cuz this is where RNA polymerase binds) to activate the liklihood of RNA polymerase transcribing —> Activator
SCENARIO 1:
- High glucose means low CAMP, so no binding to CAP and no activation of CAP and no binding to promoter and activation of transcription
- High Lactose means allolactose binds to active lac repressor protein to inactivate it and start transcription —> BUT BECAUSE CAP ISN’T ACTIVE THE EXPRESSION LEVEL IS REALLY LOW
> CAP binding structure:
- it binds to promoter region of DNA as a dimer with cAMP bound to each monomer
- it contacts the RNA polymerase to make it more likely that lac operon will be transcribed
Cute summary:
1. Glucose high, camp low, lactose absent = no expression
2. Glucose low, camp high, lactose absent = no expression - cuz the key lactose transcription influencer isnt present - allolactose
3. Glucose high, cAMP low, lactose present = low gene expression
4. Glucose low, cAMP high, lactose present = High gene expression
–> If there is lactose then repressor isnt active = promotes transcription
—> If glucose is absent then high cAMP levels = CAP binds to promoter = promoters transcription
—> If CAP binds but gene is repressed from lac repressor = no expression (CAP is just activator but lac repressor is main influencer if transcription even occurs)
—> If CAP doesnt bind and lac repressor inactive = low expression
- Explain why tight regulation of gene expression is important for eukaryotes
- In vertebrates there are many different tissue types but every single cell has the same DNA
- Different tissue cells have different structure and function due to regulation of gene expression
- Specific cells will express specific genes to carry out a specific function
~12% of our genes produce transcription factors
- Describe the basic process for the regulation of transcriptional initiation in eukaryotes
EUKARYOTES HAVE:
- seperation of transcription and translation
- Chromatin can block RNA polymerase access
- A lot of proteins are bound to chromatin and DNA - so stucture of chromatin determines what gene can and cannot be expressed
- basal transcription is low - so there is a low expression of most genes
- Majority of regulation is positive (activators) not negative (repressors) like in prokaryotes
- More proteins involved in transcriptional regulation
- More transcription factors that control each gene —> ~6 binding sites (combinatoral control)
- Describe the role of chromatin modification factors, and Mediator protein complex, in transcriptional regulation in eukaryotes
Chromatin Regulation:
- methylation of DNA and histones causes nucleosomes to pack tightly together
- transcription factors cannot bind the DNA and genes are not expressed
- Histone acetylation results in loose packing of nucleosomes
- So transcription factors bind the DNA and genes are expressed
Eukaryotic Transcriptional Regulation:
- +1 is upstream and present in mRNA
- TATA box is binding site of transcription factors
- Regulatory Elements = DNA sequence that transcription factors bind to
1. Enhancers
2. Silencers
- Transcription factors:
1. Activators
2. Repressors (rarely)
Enhancers are the DNA sequence that Activator Transcriptional Factors bind to and vice versa
> Regulation of Trancriptional Initiation:
1. Activator protein binds to enhancer site (UAS)
2. Histone modification/nucleosome remodelling complexes bind
3. Co-activator such as mediator binds and facilitates binding of TBP and first of the transcription factors
- mediator mediates between activator or repressor and general transcription factors
4. Other transcription factors TFIIB AND TFIIH bind and RNA pol II binds
5. CTD of RNA pol II is phorphorylated and transcription initiation begins
- This is why binding of activator make transcription more likely
> You can have regulatory elements anywhere, and all of these transcription factors which bind to regulatory sites interact with mediators
- There can be a large distance between basal promoter and regulatory sequences: the DNA in between these will bend —> there are proteins that will make this bend so that these activators can come into contact with mediator
SUMMARY:
Activators increase the chances and speed of RNA Polymerase II starting transcription by:
- Opening chromatin
- Bridging distant enhancers to the promoter
- Recruiting Mediator complex that recruits transcription machinery
- Helping activate RNA Pol II
- Explain how a DNA binding protein can recognise specific DNA sequences
- DNA is not an evenly rotated helix
- You have a deeper cleft and a shallower cleft = called the major and minor groove respectively
- Proteins are going to bind to DNA and transcription factors will bind DNA at specific sequences
What is available as info for the proteins to bind to is the structures on the sides of the base pairs:
- If a protein binds to the side of the minor grooves theres only 2 different types of patterns, so there is not enough info for most transcription factors to bind
- If a protein binds to the major groove, there a 4 distinct patterns for transcription factors to bind
E.g
- A DNA-binding protein (e.g. transcription factor) uses its asparagine residues to form bonds with adenine without disrupting base pair —> it can identify and bind with correct DNA sequence to carry its function
