How are differences between cells brought about when they all contain the same genes?
Differential gene expression - only a fraction of the genes in the cell are expressed
What is the most important change in a cell in a diseased state?
Expresses different set of genes
What causes cells to change gene expression?
In response to signals and other cues in the environment
What regulates the level of expression of a gene in the cell?
The level of the transcript, regulated by transcription factors
(Lots of RNA –> lots of proteins)
What charge do DNA binding proteins have?
Positive (as DNA is negatively charged)
Where do DNA biding proteins reach into on DNA?
The major groove
What are the 3 positively charge amino acids which help DNA binding proteins to be attracted to DNA?
- Arginine (Arg)
- Leucine (Leu)
- Histadine (His)
Where do GENERAL transcription factors bind to DNA?
Response element - short stretch of DNA within a promoter
Out of DNA and proteins, which has a limited topology?
How do GENERAL transcription factors recognise response elements in promoter?
- Proteins reach into the major groove and interact with groups which decorate the outside of the double helix, is a specific pattern
- H-bond acceptors and H-bond donors, as well as methyl groups (hydrophobic)
- Amino acid side chains from a-helice/b-sheet on the TF dangle into the major groove and make contacts with he DNA
- Can form H bonds with BASES
- Binds to many places on the DNA to hold the transcription factors down
What group in DNA is hydrophobic?
Methyl group CH3
What is needed to stabilise the transcription factor on the DNA?
Multiple connections between the bases on DNA and the amino acids on the transcription factor
What is Rox1 and where does it bind?
Rox1 is a transcription factor from yeast
Binds to 8 DNA sites in 3 different yeast GENES
Where are the binding sites for Rox1?
3 sites in HEM13 gene
4 sites in ANB1 gene
1 sites in ROX1 gene
Why does Rox1 transcription factor bind to its own gene?
So it can regulate itself
Negative feedback loop
What is the structure of the Rox1 binding site in genes?
All are different but all contain GTT in the centre of the site
What are 3 ways to compare the binding sequences of a transcription factor?
1) Compare the frequency at each position
2) Make a consensus sequence
3) Make a sequence logo
What is a consensus sequence?
A way of comparing the biding sequence of sites for a specific transcription factor:
- Write a sequence with the most COMMON base at each position
- Where unsure - put a different letter
What is a sequence logo?
A way of comparing the biding sequence of sites for a specific transcription factor:
- Draw the letters of the bases different sizes, depending on how common they are at that position
What can a consensus sequence be used for? (2 things)
1) To identify more genes regulated by the transcription factor
2) Identification of the sequence in other organisms and cells - homologues
Describe the affinity of the DNA biding sites for a transcription factor
Have DIFFERENT affinities, but when the transcription factor increases to a certain level - all become saturated
Site with the highest affinity is the FIRST one that transcription factor binds to - will also be the site which is occupied most of the time
However, binding is TRANSIENT
Why is the binding between a transcription factor and the binding site on DNA not perfect?
Ideal to have TRANSIENT binding to be able to MODULATE the levels of gene expression within the cells
In the DNA sequence, what is conserved between closely related species?
- Coding sequence (exons)
- Regulatory sequences in non-coding regions (introns)
What did alignment of the genes in closely related species identify?
Conserved sequences in introns, which are important regulatory sequences
Proteins which bind to these sequences
What are motifs which allow proteins (such as transcription factors, activators and repressors) to BIND TO DNA?
(4 of them)
2) Zinc fingers
3) Leucine zippers
What is the structure of a helix-TURN-helix?
- RECOGNITON helix - inserts into the major groove and makes specific contacts with the DNA with its amino acids
- Other - stabilises the position on DNA
How does a helix-TURN-helix motif bind to DNA?
As DIMERS to 2 consecutive major grooves on DNA
What is the structure of the DNA binding sequence which helix-TURN-helix bind to?
Why do transcription factors have multiple binding motifs?
To bind to DNA in many places
How does a zinc finger domain interact with DNA?
Alpha helice - recognises 2 bases in the major groove
Wraps around the DNA
Uses arginine and histine to interact with the bases on DNA (form H bonds)
What is the structure of leucine zippers?
Alpha helical monomers held together by hydrophobic amino acids to make a DIMER
Dimer forms a ‘clothes peg’ shape and interacts with 2 positions in the major groove
How can leucine zippers dimerise?
What does each type bind?
With themselves - homodimers
(Can bind symmetrical sequences)
With different leucine zippers - heterodimers
(Can bind to non-identical sequences)
What is the helix-LOOP-helix a modification of and how?
A leucine zipper
Has a loop in the structure
What does the helix-LOOP-helix structure allow?
Flexibility in how they orient in DNA
How can helix-LOOP-helix dimerise?
Why do many transcription factors bind as dimers?
Dramatically increases binding strength - synergy (combine to produce an affect greater than the sum of their separate effects)
What is the structure of a transcription factor? (what modules (4))
1) DNA binding domain
2) Protein binding domain
3) Regulatory domain
4) Activation domain
What does the protein binding domain of a transcription factor allow?
Dimerisation of the transcription factors
What does the activation domain of a transcription factor allow?
Interaction with the TRANSCRIPTION INITIATION COMPEX (RNA pol II)
What can be used to identify transcription factors which bind to a specific stretch of DNA?
What do both of these methods use?
EMSA (electrophoretic mobility shift assay)
DNAse I footprinting
BOTH involve gel electrophoresis
What is the process of EMSA?
1) Start with a sequence which contains the regulatory region (binding site)
2) Radioactively label one end of the DNA using 32P
3) Mix the DNA with cell extract (or purified transcription factor) and incubate
4) Run samples by gel electrophoresis
5) Expose the gel to identify radioactively labelled DNA
In ESMA, what cell extract is the radioactively labelled DNA mixed with?
Purified proteins from the cells which are thought to regulate the gene (where the gene is expressed)
In gel electrophoresis, what molecules travel faster in the gel?
In ESMA, when exposing the gel to show the radioactively labelled DNA, what pattern is discovered and why?
- Most of the radioactively labelled DNA is located at the bottom of the gel (no TF bound, smaller, migrates through the gel quicker)
- In some samples, there is a band of radioactivity further up on the gel (migrated slower)
- Suggests have transcription factor bound which retards migration through the gel
What is the process of DNAse I footprinting?
1) Radioactively label one end of DNA using 32P
2) Mix with cell extract (or purified transcription factor) - some proteins will bind to the DNA
3) Add DNase in very low concentrations - partially digest the DNA
4) Run the sample on a gel using gel electrophoresis- identify what TF binds to the DNA sequence
What is a probe?
Radioactively labelled portion of DNA
What does the gel look like after running gel electrophoresis in DNAse I footprinting?
Ladder-like effect where the position on the ladder represents the size of the sample
In DNAase I footprinting, what happens if the cell extract is not added and why?
Bands of MANY sizes, all the way through the gel - TF doesn’t protect the DNA binding site from being cut
If cut close to the probe - small fragment at the bottom of the gel
If cut far from the probe - long fragment near the top of the gel
In DNAase I footprinting, what happens if the cell extract added contains a TF which binds to the DNA biding site and why?
Window where there is no bands of a certain size formed, due to physical blockage of the DNAse to these sites by the transcription factor
In DNAse I footprinting, what does heat do?
Destroys the DNAse and released the binding proteins
What are PERMISSIVE transcription factors?
General Transcription factors!!
Transcription factors which DO NOT regulate gene expression but are necessary for all transcription as they bind at the promoter or interact with the TIC to initiate transcription
What are SPECIFIC or REGULATORY transcription factors?
Bind anywhere around the gene (upstream/downstream of promoter) to regulate (increase or decrease) the transcription of a gene
Bind to enhancers or silencers
Or to a REGULATORY COMPLEX
What is special about some specific/regulatory transcription factors?
They can act as BOTH an activator and a repressor
How do regulatory transcription factors regulate DNA transcription? (3 ways)
1) Interact with the RNA pol II complex
2) Alter acetylation of DNA
3) Bind to other transcription factors to form complexes
Where do regulatory TFs bind and how is this different to permissive TF?
Regulatory bind anywhere around the gene (upstream or downstream of the promoter)
- Bind to enhancers, silencers, or to a regulatory complex
Permissive only bind at promotors or to TIC
Why must DNA loop?
To allow the interaction of distant binding proteins (regulatory proteins) which must physically interact with the transcription complex
What distance away should proteins be in order for them to be able to interact and why?
As it is hard for chromatin to bend
After this, chances of finding the binding site is lower
Which genes do TF bound to enhancers activate?
Any gene within a region that they are bound to - not specific (they are promiscuous)
What are enhancers?
Binding sites on DNA for transcriptional ACTIVATORS
What are silencers?
Biding sites on DNA for transcriptional REPRESSORS
What 2 things can block the promiscuous action of transcriptional activators?
1) Insulator elements
2) Barrier sequences
Block the activator from activating genes inappropriately
How is the transcription of a single gene modulated?
1) Complexes (strongly activating/inhibiting, weakly activating/inhibiting) receive inputs from within the cell and from outside the cell
2) All inputs read by a DNA code - all have influence at the promoter about HOW MUCH the gene should be expressed
3) Combination of these inputs determine if the gene is expressed
- INPUTS are referred to as ‘genetic switches’
- Each genetic switch responds to intrinsic and extrinsic regulation
- Genetic switches work together to achieve an output
Describe tryptophan as a genetic switch
Low levels of tryptophan:
- Tryptophan genes turned on
- Tryptophan binds to TF with regulates tryptophan synthesis - represses the transcription synthesis genes
What are 7 ways to regulate a transcription factor in eukaryotes?
1) Protein synthesis
2) Ligand binding
3) Protein phosphorylation
4) Addition of a second subunit (activation subunit)
5) Unmasking (remove inhibitory protein)
6) Stimulation of nuclear entry (remove inhibitory protein)
7) Release from the membrane (cleavage)
How might one TF help another?
- Make a dimer
- Prevent ‘falling off’ the DNA
- Binding of one TF to DNA may allow the binding of another TF
How might the binding of one TF to DNA allow a different TF to bind?
The first TF may unwind the DNA slightly to reveal another binding site
What is a ‘logical network’?
What does it show?
A mathematical model of the interactions between transcription factors
Shows that depending on what TF work together - TFs work together to give an output
What 3 ways might TF work together to activate/inbhibit others?
1) Positive feedback (upregulate itself)
2) Negative feedback (downregulate itself)
3) Flip-flop (2 TF inhibit eachother)
4) Feed-forward loop (activate a chain of TFs)