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Control of Eukaryotic Gene expression > Transcriptional control of chromatin (L1-4) > Flashcards

Transcriptional control of chromatin (L1-4) Flashcards

1
Q

What is a promotor?

A

Cis acting DNA regulatory element that controls the initiation and control of transcription.

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2
Q

What is the strongest bacterial promotor ?

A

Consensus Sequence (-10 TATAAAT - -35 TTGACA)

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3
Q

What are CpG islands role in eukaryotic transcription?

A

Promotor elements eg TATA Box and initiator (Inr) are needed to initiate and control transcription in eukaryotes. However, many mammals lack these promotor regions or they are just not obvious, so CpG islands are needed. C residues in CpG islands escape methylation (become hypomethylated). Hypomethylation is associated with initiation of transcription.

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4
Q

What is methylation of CpG islands and what is it associated with?

A

When c residues followed by a G are methylated. Methylation causes transcription to be turned off. Therefore methylation is a negative thing for transcription in eukaryotes.

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5
Q

What are the regulatory regions on a eukaryotic gene?

A

Areas which turn transcription on or off.
>UAS and enhancers have binding sites for activator proteins, therefore activate transcription.
>URS and silencers have repressor binding sites.

UAS and URS are closer to transcription start site.

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6
Q

Reporter analysis is a powerful tool used for studying promotor action. What specific reporter gene was used to study what promotor in a mouse embryo?

A

Lac Z reporter used to study ure2 promoter. The more Lacz present will show how the promotor is working. Would be a linear relationship.

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7
Q

How many RNA Polymerases do prokaryotes and eukaryotes have?

A

P- 1
E- 3 (RNA POl 2 most important in transcription)

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8
Q

Describe the process of formation of pre initiation complex in eukaryotes.

A
  1. TFIID binds to TATA region of promotor. TFIID involves TBP and TAFs. Recruits TFIIB and TFIIA.
  2. TFIIA stabilises binding of TFIID. TFIIB recruits RNA POL 2 which is associated with TFIIF (helps RNA POL 2 bind).
  3. TFIIF simulates elongation and destablises non specific RNA POL 2 - DNA interactions.
  4. TFIIE recruits TFIIH.
  5. TFIIH is very important, see further card.
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9
Q

What subunits make up TFIID?
(first transcription factor with 13 subunits and has a trilobular structure)

A

TBP (TATA binding protein) and TAFs (TBP associated factors).

TBP is shaped like a molecular saddle.
TAFs promote interaction of TFIID with basal promotors and interact with activators.

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10
Q

The final transcription factor is TFIIH. What are the properties?

A
  1. TFIIH has ATPase activity which is required for promoter melting. TFIIH uses this energy to act as a helices to separate the strands. The ATPase is called XPB (or Ssl2)
  2. The CAK part of the factor contains the kinases which phosphorylate the C terminal domains of the RNA POL (CTD). CTD domain is phosphorylated whilst pol 2 starts transcribing.. This is promotor clearence.
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11
Q

what is the ATPase called in TFIIH?

A

XPB or Ssl2

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12
Q

Where are the C terminal domains located on the RNA POL?

A

The RPB1 subunit which is homologous to B’ on prokaryotic RNA Pol.

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13
Q

UAS/enhancers are used to increase transcription levels. What are the two classes of UAS/enhances

A

1.Common sequence elements- common means constant activation of promotors. These are located close to core promoter and bind activators that are abundant and constantly active.
2. response elements - bind factors whose activity is induced by stimuli.

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14
Q

What are 3 examples of ‘Common’ sequence elements and what factors bind to them?

A

1.GC Box - binds SP1 factor
2. Octamer - binds Oct-1 factor
3. CAAT Box - Binds NFY factor.

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15
Q

What are two examples of response elements, what do they bind and what is the factor induced by?

A

SRE- binds serum response factor which is induced by growth factors
HSE - binds heat shock factor which is induced by heat shock

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16
Q

what does it mean that eukaryotic activators are MODULAR?

A

The activation domain, DNA binding domain and flexible protein domains are independent units. They are separate. The activation

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17
Q

Activation domains are one of the domains in activators. Activation domains are characterised due to their amino acid composition. What are 3 examples of activation domains?

A
  1. Acidic patch - clusters of negative charged residues (Asp/glu) eg VP16
  2. Glutamine rich eg Sp1
  3. Proline rich eg Jun
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18
Q

What 2 main domains are activator proteins made up of?

A

Activation domain and DNA binding domain.

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19
Q

Two ways to analyse promotor regions?

A

Sequence comparisons and reporter analysis.

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20
Q

What do activators do- 3 different mechanisms?

A
  1. promote binding of additional activators
  2. increase rate of PIC formation
  3. stimulate release of stalled RNA pol 2.
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21
Q

How do activators increase rate of PIC formation?

A
  • increase TFIID binding
  • increase TFIIB binding
  • increase RNA Pol 2 recruitment
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22
Q

What do mediators do for activators?

A

Meditators bridge the gap between activators and RNA pol 2. Mediators are large complexes (22 polypeptides) with 3 domains (Head, middle and tail).

23
Q

What are the 4 core histones? What are the N terminal domains and globular domains made up of?

A

H2A, H2B, H3 and H4.

N terminal domain - highly basic, rich in lys and arg

Globular domain- alpha helices and loops

24
Q

What type of proteins are histones?

A

basic

25
Q

What is a nucleosome made up of ?

A

Core histones. H2A and H2B dimers and H3-H4 tetramers.

26
Q

What is an example of a linker histone and where do they sit?

A

H1 histone. They bind to the DNA between the nucleosomes, and also can result in thicker fibres (30nm).

27
Q

Chromatin inhibits transcription. What evidence proves this?

A
  1. In vitro experiments - when naked DNA is present, transcription happens. When chromatin is present, transcription is stopped.
  2. In vivo experiments- shows that nucleosomes are lost during transcription which shows that chromatin is not present.
  3. Studies of Saccharomyces cerevisiae- GAL4 promotor is turned on when galactose is present. This increases expression of H4 and therefore increases chromatin, decreasing transcription. When glucose is present, GAL4 promotor is turned off, H4 expression decreases and transcription increases.
    (OVERALL, GLUCOSE INCREASES TRANSCRIPTION)
28
Q

What are 3 mechanics for modulating chromatin structure?

A
  1. Histone variants
  2. Post translational modification of histones.
  3. ATP dependent chromatin remodelling.
29
Q

Histone variants are one way of modulating chromatin structure in order to increase transcription. What are some examples of histone variants?

A

H2A - H2AZ, H2AX, macroH2A

H2B - hTSH2B

H3- CENP-A, H3.3

30
Q

What are the 4 ways to post translationally modify histones?

A
  1. Acylation
  2. Methylation
  3. Ubiquitylation
  4. Phosphorylation
31
Q

What are the two enzymes involved in histone lysine acylation?

A

Histone acetyl transferases (HATs) adds acetyl group to lysines.

Histone Deacetylases (HDACs) remove acetyl group.

32
Q

Acetylation increases transcriptional activation. What evidence proves this?

A

First nuclear HAT discovered was homologous to yeast Gcn5, which is a transcriptional activator.

33
Q

What are the two main types of histone acetyl transferases (HATs)?

A
  1. GNAT family
  2. MYST family
34
Q

How do activators interact with HATs?

A
  1. HATs contain subunit that interacts with activators eg Tra1 (yeast) and TRRAP (mammals)
  2. Some HATs are actually already in the transcription machinery.
35
Q

What are the two ways that acetylation increases transcriptional activation?

A
  1. Acetylation reduces negative charge of chromatin, so N terminals cannot bind as tightly, so chromatin opens up and transcription can occur.
  2. recruitment of bromodomain proteins which increase transcription. Acelylated lysine residues are recognised by bromodomains.
36
Q

When bromodomains bind to histones transcription is increased. What two examples of bromodomains?

A
  1. Bdf1 - binds to acelyated H4 and recruits TFIID
  2. TAFII250 - subunit of TFIID and bind to acetylated H4.
37
Q

Methylation is another way of post translationally modifying histones. What enzymes are involved in methylation of histones?

A

1.Histone- lysine methy transferases (HKMTs). These can add 1, 2 or 3 methyl groups to lysines.

  1. Lysine demethylases- these remove the lysines.
38
Q

Does methylation affect greatly chromatin structure?
Is methylation readily reversible like aceylation?

A

No. As methylation does not affect chromatin charge, it doesn’t have much effect on structure.

No, it is not readily reversible.

39
Q

Methylation of histones can either activate or repress transcription. Which methyl- lysine residues activate and repress?

A

H3 Lys9 and H3 lys27 REPRESS

(9 and 27 and multiples and sound like harsher numbers therefore repress)

H3 Lys4 and H3 lys34 ACTIVATE

40
Q

What do bromodomains and chromodomains recognise?

A

Bromodomains - acetylated lysine residues
Chromodomains - methylated lysine residues

41
Q

Nucleosomes are dynamic so they can move and allow chromatin to be remodelled, which increases transcription. What is chromatin remodelling?

A

When the nucleosomes in chromatin slide, unwrap, space out, removed, histone variant exchange etc. this will enable activator binding sites and promotor regions to be exposed so that transcription can occur.

42
Q

SWI/SNF is important in ATP dependent chromatin remodelling. What is it and what does it do?

A

SWI/SNF is a complex with a catalytic subunit Snf2 which, in the presence of ATP, causes the DNA to slide over the nucleosome, exposing various binding sites on the DNA.

43
Q

How does SWI/SNF and Gcn5 HAT cooperatively activate transcription?

A

Gcn5 HAT acetylates lysine residues in histones and causes them to be less negative and therefore the nucleosome is less tightly wrapped, so transcription is increased. SWI2/SNF2 subfamily of SWI/SNF has a bromodomain which is attracted towards acetylated lysine residues. This causes the nucleosome to slide and expose more binding sites, further activating transcription.

44
Q

What do all the SWI/SNF ATPase superfamily have?

A

Snf2-related ATPase

45
Q

Which SWI/SNF subfamily has a chromodomain? And what would this recognise?

A

CHD/Mi2 subfamily. Chromodomains recognise methylated lysine residues.

46
Q

What are the 3 ways to repress transcription?

A
  1. Histone Deacetylases (HDACs)
  2. ATP-dependent remodellers - some are activators and some are repressors
  3. Histone methylases - H3Lys9 and 27 are the repressor ones
47
Q

Histone deacetylases (HDACs) are transcriptional repressors. They remove acetyl groups from lysine. What are the 4 groups?

A

> Class 1, 2 and 4 are classical HDACs (zinc dependent)
Class 3 are Sir2 family (SIRTUINS) and require NAD as a cofactor.

48
Q

Histone deacetylases (HDACs) act as co- repressors with what?

A

Sin3

49
Q

Some ATP-dependent chromatin remodellers repress transcription. What is an example of one which represses?

A

Nurd Complex which belongs to Mi2/CHD (contains chromodomain which recognises methylated lysine) complex. When Nurd is present, transcription is turned off.

50
Q

What are 3 features of heterochromatin? (remember heterochromatin is associated with transcriptional repression).

A
  1. Hypoacetylation - low levels of transcription
  2. H3 methylation on lys9 or lys27
  3. Association with specific silencing factors
51
Q

What is Suvar39?

A

an example of a histone lysine methyl transferase (HKMTs). Adds methyl group to lysine 9 causing transcriptional repression.

52
Q

what is heterochromatin protein 1 ?

A

a chromodomain protein specific to H3 Lys9me. It compacts the nucleosome and prevents recruitment of RNA pol. It causes transcriptional repression.

53
Q

How does X chromosome inactivation occur?

A

Tsix gene is turned off.
Xist is unregulated and it coats the chromosome, then H3lys27me is recruited with also recruits chromodomain heterochomatin, causing further transcriptional repression. Barr body created.

Control of Eukaryotic Gene expression (3 decks)

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