Lecture 10 Flashcards
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.
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
Rox1 is a DNA binding protein found in yeast, how many different sites does it bind to
8 sites in three different yeast genes
Transcription factors recognise short stretches of DNA through interactions with individual base pairs, 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
Usually the consensus sequence is the sequence with the highest affinity for the DNA binding protein, T or F
F – often it isn’t
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
Give an example of a permissive transcription factor
TATA binding protein (TBP)
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.
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
The Rox1 gene contains a binding site for the Rox1 protein itself, what is the significance of this
Rox1 regulates its own transcription
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
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
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 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
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 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 type of bonding holds DNA binding proteins in place at the major groove of the DNA
hydrogen bonds
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
Cells often express different genes in a diseased state, T or F
T
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
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 is the name given to the sequence in the DNA to which transcriptional repressors bind
Silencers
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
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
Which sequence is perfectly conserved across all Rox1 binding sites
GTT
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
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 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
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
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.
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
