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BMS242 Cell and Molecular > L9 - Control of Gene Expression > Flashcards

L9 - Control of Gene Expression Flashcards

1
Q

What is different with different cells and what is the same

A

Same genes in all cells but in different cells - different genes expressed - different proteins produced

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2
Q

What is expressed when cells are in a diseased state

A

Different genes expressed

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3
Q

What does the level of transcription determine

A

Level of expression

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4
Q

High transcription ==>

A

Lots of mRNA - high level of expression - increased level of proteins

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5
Q

Two properties of all DNA bidning proteins

A

Positively charged

Reach into the major groove

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6
Q

What are common positively charged amino acids

A

Arginine and lysine

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7
Q

The binding site for DNA binding proteins AKA

A

Respone element

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8
Q

Why is it so hard for TFs to bind to DNA

A

Because DNA has limitted topology

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9
Q

Interactions with G are possible due to …

A

Two hydrogen bond acceptors

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10
Q

Interactions with A are possible due to …

A

A hydrogen bond acceptor and a hydrogen bond donor

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11
Q

Interactions with C are possible due to …

A

Hydrogen atom and a hydrogen bond donor

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12
Q

Interactions with T are possible due to …

A

Methyl group and a hydrogen bond acceptor

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13
Q

What is a common AA that is able to form H bonds with ___ (base)

A

Asparagine with adenine (A)

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14
Q

Rox 1 binds to __ sites within ___ genes

A

8 sites in 3 genes

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15
Q

What is a consensus sequence

A

the sequence in which the amino acid positions are represented by the most commonly occuring amino acid at those positions

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16
Q

How does a sequence logo confer consensus sequence

A

Height of each letter proportional to the frequency the AA is found at

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17
Q

What is the consensus sequence for Rox1

A

YCHATTGTTCTC

Where Y = C/T and H = A/C/T

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18
Q

What can be said about the different sites

A

They have different affinities for the proteins

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19
Q

How do non coding regions change throughout evolution

A

Change rapidly as they serve no function so there is no selection pressure acting on them

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20
Q

How have coding regions changed througout evolution

A

Highly conserved

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21
Q

How have regulatory regions changed throughout evolution

A

Highly conerved

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22
Q

What are the four DNA binding motifs

A

Helix turn helix
Zn finger
Leucine zipper
Helix loop helix

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23
Q

What is the helix turn helix made from

A

Two helicies

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24
Q

Describe how the helix turn helix binds to the DNA

A

Recognition helix inserts into the major groove to make specific contacts

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25
Q

How do helix - turn - helix tend to bind

A

In dimers at two consecutive major grooves

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26
Q

What can be said about the recognition sequence

A

Palindromic so the the dimers form head to head

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27
Q

How many AAs hold the Zn atom

A

Four

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28
Q

How does the Zn finger bind to DNA

A

a-helix interacts with the major groove - recognises two bases and usually binds in multiples

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29
Q

What is the structure of the leucine zipper

A

Two a helix monomers held by hydrophobic amino acids (leucine) forming a dimeric strucutre

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30
Q

Leucine zipper homodimers

A

Made of the same monomers – binds identical sequences

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31
Q

Leucine zipper heterodimers

A

Made of different monomers - binds different sequences

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32
Q

What is the helix loop helix related to

What is one way that it is different

A

Leucine zipper

But loop for added flexibility

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33
Q

What property of DNA binding proteins increases the binding strength

A

Co-operative binding

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34
Q

TFs are modular - what domains do they contain

A

DNA binding
Reg
Act/rep
Protein binding

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35
Q

Two ways of identifying DNA binding proteins

A

EMSA

DNAse I footprint

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36
Q

What does EMSA stand for

A

Electrophoretic mobility shift assay

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37
Q

Explain the steps required for EMSA

A

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

38
Q

Describe how a DNAse I footprint may be carried out

A

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

39
Q

What can a DNAse footprint also be used to determine

A

WHERE a protein binds to on a specific length of DNA

40
Q

What are permissive TFs

A

AKA general TFS
Required for all transcription
Binds at the promoter

41
Q

What are specific/regulatory TFS

A

ACTIVATORS - Increase transcription by neighbouring genes

REPRESSORS - Reduce transcription at neighbouring genes

42
Q

How do regualtory transcription factors function

A

Interact with the RNA polymerase complex
Alter acetylation of the DNA
Reg binding of other TFs

43
Q

Where do regulatory TFs bind

A

Anywhere around the gene - or don’t bind to the DNA directly but bind to the mediator complex

44
Q

What is the role of DNA looping

A

Chromatin does not bend easily so for two proteins to interact they need to be
At neighbouring sequences or over 500 bp apart

45
Q

What stops regulatory sequences affecting other genes

A

Insulators and barriers

46
Q

What is an enhancer

A

Binding site for transcriptional activators

47
Q

What is a silencer

A

Binding site for represors

48
Q

Enchances are promisousos

What does this mean

A

They will work on any gene

49
Q

What is an insulator

A

Prevent effect on neighbouring genes

50
Q

What is a barrier

A

Prevent any effects of heterochromatin

51
Q

What inputs alter gene expression

2 act) (1 inhib

A

ACT strongly activating assembly
ACT weakly activating protein
INHIB Strongly inhibbiting protein

52
Q

What are the inputs which alter gene expression AKA

A

Genetic switches

Respond to intrinsic and extrinsic signals

53
Q

Desribe the action of the tryptophan repressor

A

trp repressor protein represses the genes required for the synthesis of tryptophan

54
Q

Ways which TFS can be regualted and go from inactive to active

A 
Protein synthesis 
Ligand binding 
Protein phosphorylation 
Add of a 2nd subunit 
Unmasking 
Stim of nuclear entry 
Release from the membrane
55
Q

Four ways TFS can regualted exp of other TFs

A

Positive feedback loop
Negative feedback loop
Flip flop device
Feed forward loop

56
Q

How does synergistic action aid TF binding

A

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

57
Q

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

A

T

58
Q

Which groove of the DNA do protein interact with and why

A

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

59
Q

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

A

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

60
Q

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

A

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

61
Q

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

A

T

62
Q

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

A

hydrogen bonds

63
Q

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

A

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

64
Q

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

A

8 sites in three different yeast genes

65
Q

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

A

Rox1 regulates its own transcription

66
Q

Which sequence is perfectly conserved across all Rox1 binding sites

A

GTT

67
Q

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

A

T

68
Q

What is meant by the consensus sequence

A

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.

69
Q

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

A

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

70
Q

What is meant by a sequence logo

A

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

71
Q

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

A

F – often it isn’t

72
Q

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

A

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

73
Q

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

A

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

74
Q

Describe the structure and interaction of leucine zipper domains with DNA

A

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

75
Q

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

A

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.

76
Q

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

A

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

77
Q

What is meant by the term permissive transcription factors

A

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

78
Q

Give an example of a permissive transcription factor

A

TATA binding protein (TBP)

79
Q

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

A

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.

80
Q

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

A

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

81
Q

How do regulatory transcription factors function

A

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

82
Q

What is meant by DNA looping

A

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

83
Q

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

A

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

84
Q

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

A

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

85
Q

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

A

Silencers

86
Q

What is meant by the probability of transcription

A

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.

87
Q

Give an example of a simple genetic switch

A

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

88
Q

Recall the four hypothesised mechanisms of regulating transcription factors

A

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

89
Q

What is meant by the term transcription factor synergy

A

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

90
Q

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

A

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

BMS242 Cell and Molecular (26 decks)

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