Lecture 10

Question 1

How do specific or regulatory transcription factors differ from permissive transcription factors

Answer 1

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.

Question 2

What is meant by the term transcription factor synergy

Answer 2

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

Question 3

Rox1 is a DNA binding protein found in yeast, how many different sites does it bind to

Answer 3

8 sites in three different yeast genes

Question 4

Transcription factors recognise short stretches of DNA through interactions with individual base pairs, T or F

Answer 4

T

Question 5

Which groove of the DNA do protein interact with and why

Answer 5

DNA binding proteins interact with the major groove of the DNA because the minor groove is too narrow

Question 6

What is significant and unusual about DNA binding proteins that allows them to bind to DNA

Answer 6

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

Question 7

Usually the consensus sequence is the sequence with the highest affinity for the DNA binding protein, T or F

Answer 7

F – often it isn’t

Question 8

What is meant by DNA looping

Answer 8

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

Question 9

Give an example of a permissive transcription factor

Answer 9

TATA binding protein (TBP)

Question 10

What is meant by the probability of transcription

Answer 10

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.

Question 11

Explain how electrophoretic mobility shift assays can be used to identify DNA binding proteins

Answer 11

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

Question 12

What is meant by the term permissive transcription factors

Answer 12

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

Question 13

The Rox1 gene contains a binding site for the Rox1 protein itself, what is the significance of this

Answer 13

Rox1 regulates its own transcription

Question 14

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

Answer 14

T

Question 15

Describe the structure and interaction of leucine zipper domains with DNA

Answer 15

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

Question 16

Recall the four hypothesised mechanisms of regulating transcription factors

Answer 16

Protein synthesis of inhibitors etc. Ligand binding, protein phosphorylation and addition of subunits

Question 17

What is meant by a sequence logo

Answer 17

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

Question 18

How do regulatory transcription factors function

Answer 18

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

Question 19

What is the role of insulators and barriers in the control of gene expression

Answer 19

Insulators and barriers block regulatory sequences from affecting neighbouring genes and prevent enhancers from activating the wrong genes

Question 20

What type of bonding holds DNA binding proteins in place at the major groove of the DNA

Answer 20

hydrogen bonds

Question 21

What do Y, H and N represent in the consensus sequences

Answer 21

Y represents either a C or a T. H represents and A, C or T. N refers to any base

Question 22

Cells often express different genes in a diseased state, T or F

Answer 22

T

Question 23

Specific/regulatory transcription factors don’t have to bind directly to the gene which they regulate, T or F

Answer 23

T – they can exert their regulation of transcription by binding to a regulatory complex

Question 24

What is meant by the consensus sequence

Answer 24

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.

Question 25

What is the name given to the sequence in the DNA to which transcriptional repressors bind

Answer 25

Silencers

Question 26

Describe the structure and interaction of zinc finger domains with the DNA

Answer 26

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

Question 27

DNA binding proteins form stable interactions with the DNA sequence to which they bind, T or F

Answer 27

F – the interactions between DNA and DNA binding proteins are not stable

Question 28

Which sequence is perfectly conserved across all Rox1 binding sites

Answer 28

GTT

Question 29

What attribute of enhancer sequences results in the need of insulators and barriers

Answer 29

Enhancers are binding sites for transcriptional activators. These sequences are usually promiscuous and activate transcription of any adjacent genes. Insulators and barriers are required

Question 30

Give an example of a simple genetic switch

Answer 30

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

Question 31

Recall the four logic-based mechanisms by which transcription factors can regulate the transcription of other transcription factors

Answer 31

Positive feedback, negative feedback, flip-flop devices and feedforward mechanisms

Question 32

Like proteins, DNA has unlimited topology and can adopt many shapes, T or F

Answer 32

F – DNA has a limited topology and defined 3D structures due to set interactions between the 4 bases following certain rules

Question 33

Explain the process of DNAse I footprinting in the identification of DNA binding proteins

Answer 33

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.

Question 34

The helix-turn-helix domain is an example of a DNA binding domain, describe its structure and interaction with DNA

Answer 34

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