L9 - Control of Gene Expression Flashcards
What is different with different cells and what is the same
Same genes in all cells but in different cells - different genes expressed - different proteins produced
What is expressed when cells are in a diseased state
Different genes expressed
What does the level of transcription determine
Level of expression
High transcription ==>
Lots of mRNA - high level of expression - increased level of proteins
Two properties of all DNA bidning proteins
Positively charged
Reach into the major groove
What are common positively charged amino acids
Arginine and lysine
The binding site for DNA binding proteins AKA
Respone element
Why is it so hard for TFs to bind to DNA
Because DNA has limitted topology
Interactions with G are possible due to …
Two hydrogen bond acceptors
Interactions with A are possible due to …
A hydrogen bond acceptor and a hydrogen bond donor
Interactions with C are possible due to …
Hydrogen atom and a hydrogen bond donor
Interactions with T are possible due to …
Methyl group and a hydrogen bond acceptor
What is a common AA that is able to form H bonds with ___ (base)
Asparagine with adenine (A)
Rox 1 binds to __ sites within ___ genes
8 sites in 3 genes
What is a consensus sequence
the sequence in which the amino acid positions are represented by the most commonly occuring amino acid at those positions
How does a sequence logo confer consensus sequence
Height of each letter proportional to the frequency the AA is found at
What is the consensus sequence for Rox1
YCHATTGTTCTC
Where Y = C/T and H = A/C/T
What can be said about the different sites
They have different affinities for the proteins
How do non coding regions change throughout evolution
Change rapidly as they serve no function so there is no selection pressure acting on them
How have coding regions changed througout evolution
Highly conserved
How have regulatory regions changed throughout evolution
Highly conerved
What are the four DNA binding motifs
Helix turn helix
Zn finger
Leucine zipper
Helix loop helix
What is the helix turn helix made from
Two helicies
Describe how the helix turn helix binds to the DNA
Recognition helix inserts into the major groove to make specific contacts
How do helix - turn - helix tend to bind
In dimers at two consecutive major grooves
What can be said about the recognition sequence
Palindromic so the the dimers form head to head
How many AAs hold the Zn atom
Four
How does the Zn finger bind to DNA
a-helix interacts with the major groove - recognises two bases and usually binds in multiples
What is the structure of the leucine zipper
Two a helix monomers held by hydrophobic amino acids (leucine) forming a dimeric strucutre
Leucine zipper homodimers
Made of the same monomers – binds identical sequences
Leucine zipper heterodimers
Made of different monomers - binds different sequences
What is the helix loop helix related to
What is one way that it is different
Leucine zipper
But loop for added flexibility
What property of DNA binding proteins increases the binding strength
Co-operative binding
TFs are modular - what domains do they contain
DNA binding
Reg
Act/rep
Protein binding
Two ways of identifying DNA binding proteins
EMSA
DNAse I footprint
What does EMSA stand for
Electrophoretic mobility shift assay
Explain the steps required for EMSA
Radioactively label one end of the DNA (P32)
Mix with cell extract or purified (DNA binding) protein
Run samples by gel electrophoresis
DNA binding protein will retard movement through the gel (bigger sample with protein bound) so wont move as far other samples
Describe how a DNAse I footprint may be carried out
Radioactively label one end of the DNA (use 32P this is the probe)
Mix with cell extract or purified protein
Add DNAse which will randomly cut the DNA at an average of one random cut per molecule of DNA
If DNA binidng protein is bound this will protect the sample from cleavage
Heat to denature the DNAse and release the DNA biding protein
Run a gel electrophoresis
Blank region is the region where the DNA was protected from digest by the binding protein
What can a DNAse footprint also be used to determine
WHERE a protein binds to on a specific length of DNA
What are permissive TFs
AKA general TFS
Required for all transcription
Binds at the promoter
What are specific/regulatory TFS
ACTIVATORS - Increase transcription by neighbouring genes
REPRESSORS - Reduce transcription at neighbouring genes
How do regualtory transcription factors function
Interact with the RNA polymerase complex
Alter acetylation of the DNA
Reg binding of other TFs
Where do regulatory TFs bind
Anywhere around the gene - or don’t bind to the DNA directly but bind to the mediator complex
What is the role of DNA looping
Chromatin does not bend easily so for two proteins to interact they need to be
At neighbouring sequences or over 500 bp apart
What stops regulatory sequences affecting other genes
Insulators and barriers
What is an enhancer
Binding site for transcriptional activators
What is a silencer
Binding site for represors
Enchances are promisousos
What does this mean
They will work on any gene
What is an insulator
Prevent effect on neighbouring genes
What is a barrier
Prevent any effects of heterochromatin
What inputs alter gene expression
2 act) (1 inhib
ACT strongly activating assembly
ACT weakly activating protein
INHIB Strongly inhibbiting protein
What are the inputs which alter gene expression AKA
Genetic switches
Respond to intrinsic and extrinsic signals
Desribe the action of the tryptophan repressor
trp repressor protein represses the genes required for the synthesis of tryptophan
Ways which TFS can be regualted and go from inactive to active
Protein synthesis Ligand binding Protein phosphorylation Add of a 2nd subunit Unmasking Stim of nuclear entry Release from the membrane
Four ways TFS can regualted exp of other TFs
Positive feedback loop
Negative feedback loop
Flip flop device
Feed forward loop
How does synergistic action aid TF binding
Binding of one to another prevents them falling off one another
Because eah protein would need to lose two interactions to fall off
Binding of one transcription factor may aid binding of another TF
Cells often express different genes in a diseased state, T or F
T
Which groove of the DNA do protein interact with and why
DNA binding proteins interact with the major groove of the DNA because the minor groove is too narrow
What is significant and unusual about DNA binding proteins that allows them to bind to DNA
DNA binding proteins have a positive charge due to high positively charged amino acids contained within them. This allows the protein to interact and remain bound to the negatively charge phosphate backbone
Like proteins, DNA has unlimited topology and can adopt many shapes, T or F
F – DNA has a limited topology and defined 3D structures due to set interactions between the 4 bases following certain rules
Transcription factors recognise short stretches of DNA through interactions with individual base pairs, T or F
T
What type of bonding holds DNA binding proteins in place at the major groove of the DNA
hydrogen bonds
DNA binding proteins form stable interactions with the DNA sequence to which they bind, T or F
F – the interactions between DNA and DNA binding proteins are not stable
Rox1 is a DNA binding protein found in yeast, how many different sites does it bind to
8 sites in three different yeast genes
The Rox1 gene contains a binding site for the Rox1 protein itself, what is the significance of this
Rox1 regulates its own transcription
Which sequence is perfectly conserved across all Rox1 binding sites
GTT
Binding sites for the same DNA binding protein all usually show a similar frequency of bases at the same location in binding site genes, T or F
T
What is meant by the consensus sequence
The consensus sequence shows the generally conserved sequence common to all binding sites for a DNA binding protein. This can be used to identify other binding sites with the same consensus sequence and that may bind to a DNA binding protein of interest.
What do Y, H and N represent in the consensus sequences
Y represents either a C or a T. H represents and A, C or T. N refers to any base
What is meant by a sequence logo
A sequence logo is a size representation of the bases within a DNA sequence where the size of the letters represents the frequency of bases within the sequence
Usually the consensus sequence is the sequence with the highest affinity for the DNA binding protein, T or F
F – often it isn’t
The helix-turn-helix domain is an example of a DNA binding domain, describe its structure and interaction with DNA
Helix-turn-helix domains consist of 2 ?-helices, one of which, the recognition helix interacts with the major groove of the DNA by making specific contacts with bases. Helix-turn-helix domains usually bind as dimers to palindromic recognition sequences contained within two consecutive major grooves
Describe the structure and interaction of zinc finger domains with the DNA
Zinc finger domains consist of an ?-helix and B-sheet. The ?-helix interacts with the major groove of the DNA by interactions of arginine and histidine residues with bases in the DNA sequence. These domains require the presence of a Zn2+ ion to stabilise the structure and hold the domain in place. These domains are usually found in combination with several other zinc fingers
Describe the structure and interaction of leucine zipper domains with DNA
Leucine zippers consist of 2 ? helices that form a dimer held together by hydrophobic amino acids such as leucine. These domains straddle the DNA binding to symmetrical sequences in the case of homodimers, or non-identical sequences if the two helices are a heterodimer
Explain the process of DNAse I footprinting in the identification of DNA binding proteins
Once you have a known sequence of DNA that you want to discover interacting proteins for, you radioactively label it with 32P. This sequence is then mixed with a cell extract or purified proteins to allow binding of potentially interacting partners. The DNAse enzyme is then added to the mixture to partially digest the sequences by cleaving each piece only once. The sample is then heated to destroy the DNAse enzyme and release the binding proteins. The sample is then run by gel electrophoresis. If a DNA binding protein has bound, then the region of DNA where its binds will be protected from the cleavage by the DNAse enzyme. This will be represented as a missing band in the gel.
Explain how electrophoretic mobility shift assays can be used to identify DNA binding proteins
EMSA involves the radioactive labelling of a known sequence of DNA that contains the binding site to which you want to identify interacting proteins for. This labelled DNA in then mixed with purified proteins or cell extracts. Instead of adding DNAse and heating, the mixture is then immediately run by gel electrophoresis. If a protein has bound to the labelled sequence, then it won’t move as far though the gel as the unbound DNA and would be represented by an additional band in the gel
What is meant by the term permissive transcription factors
Permissive transcription factors are general transcription factors necessary for all transcription and are non-regulatory. These bind at the promoter sequence of the gene and are ubiquitously expressed. Binding of permissive transcription factors to the promoter helps the polymerase machinery to find the start site
Give an example of a permissive transcription factor
TATA binding protein (TBP)
How do specific or regulatory transcription factors differ from permissive transcription factors
Specific or regulatory transcription factors bind to specific genes are play a regulatory role in transcription. They can be transcriptional activators or transcriptional repressors and bind anywhere in the gene, sometimes quite far away.
Specific/regulatory transcription factors don’t have to bind directly to the gene which they regulate, T or F
T – they can exert their regulation of transcription by binding to a regulatory complex
How do regulatory transcription factors function
They interact with the RNA polymerase complex and either alter acetylation of the DNA (which effects chromatin structure), bind to other transcription factors or act upstream of permissive/general transcription factors
What is meant by DNA looping
Chromatin is quite stiff and so does not bend easily. It is thus thought that for two proteins to interact with the DNA they need to bind directly to neighbouring DNA sequences. These binding sequences for regulatory transcription factors need to be over 500 base pairs apart, this leads to DNA looping
What is the role of insulators and barriers in the control of gene expression
Insulators and barriers block regulatory sequences from affecting neighbouring genes and prevent enhancers from activating the wrong genes
What attribute of enhancer sequences results in the need of insulators and barriers
Enhancers are binding sites for transcriptional activators. These sequences are usually promiscuous and activate transcription of any adjacent genes. Insulators and barriers are required
What is the name given to the sequence in the DNA to which transcriptional repressors bind
Silencers
What is meant by the probability of transcription
There are usually many inputs that alter gene expression, referred to as genetic switches. Each switch responds to intrinsic or extrinsic regulation. The combination of enhancers, repressors, silencers and activators is what determines the probability of transcription.
Give an example of a simple genetic switch
The tryptophan repressor protein represses genes required for tryptophan synthesis and storage. When levels of tryptophan are high then it turns off genes required for tryptophan synthesis
Recall the four hypothesised mechanisms of regulating transcription factors
Protein synthesis of inhibitors etc. Ligand binding, protein phosphorylation and addition of subunits
What is meant by the term transcription factor synergy
Transcription factors cooperate in order to influence gene transcription. Binding of one transcription factor to another may help prevent them from falling off the DNA. Instead of one interaction, each protein would need to lose two interactions to fall off the DNA. Similarly, binding of one transcription factor to DNA may enable another transcription factor to bind to that sequence also
Recall the four logic-based mechanisms by which transcription factors can regulate the transcription of other transcription factors
Positive feedback, negative feedback, flip-flop devices and feedforward mechanisms
