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Transcription machinery Flashcards

1
Q

How many subunits does RNAP II have?

A

12

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2
Q

What is unique about RNAPII structure?

A

Contains a Carboxy Terminal domain

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3
Q

What does the CTD consist of?

A

Tandem heptapeptide repeats of the consensus sequence: tyr-ser-pro-thr-ser-pro-ser

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4
Q

How many repeats of the consensus sequence do humans contain?

A

52

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5
Q

Is there redundancy to the importance of the repeats?

A

There is evidence that yeast can delete some repeats and still be viable but if more than 13 copies of the repeat are deleted then it is lethal.

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6
Q

What is the most important post translational modification of the amino acids in the CTD? And where?

A

Phosphorylation, at serine 2 and 5.

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7
Q

Explain how CTD length is related to gene density

A

By comparison of different species the CTD length seems to be INVERSELY related to gene density within the species.

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8
Q

How can phosphorylated CTD help chromatin modification?

A

Phosphorylated CTD can recruit proteins which assist in histone methylation and regulation of histone Acetylation, methylation recruits elongation factors.

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9
Q

Is PolII recruited to promotors that have modified or unmodified CTD?

A

unmodified

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10
Q

Why is the CTD crucial for forming the PIC?

A

It has high affinity for the Mediator complex which acts as a bridge between the transcription machinery and activators

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11
Q

What is responsible for the high affinity between Mediator and CTD?

A

Hydrogen bonds and hydrophobic interactions

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12
Q

How does the CTD become phosphorylated?

A

CDK7 which is a part of TFIIH, phosphorylates Ser5 and Ser7 breaking the H bonds between RNAP and Mediator aiding promoter escape for RNAPII. After this other residues are phosphorylated and dephosphorylated by CTD kinases.

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13
Q

What is the major regulatory step in elongation?

A

Promoter-proximal pausing

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14
Q

When does RNAPII pausing occur?

A

When Negative Elongation factor (NELF) and DRB-inducing sensitivity factor ( DISF) bind.

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15
Q

Is CTD postranslationally modified in elongation?

A

yes

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16
Q

How does RNAPII pause release occur?

A

Positive transcription factor B (p-TEFb) phosphorylates NELF which removes NELF from RNAPII. Phosphorylation of DSIF converts it to a positive elongation factor leading to pause release.

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17
Q

What does p-TEFb also phosphorylate?

A

Ser2, leading to recruitment of elongation factors and chromatin modifying factors.

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18
Q

What other CTD kinases have been recently shown to also play a key role in the regulation of pause release, highlighting importance of CTD phosphorylation?

A

BRD4 and PAF1. The CTD also promotes the recruitment of PAF1.

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19
Q

What is the importance of quick RNA capping?

A

Downstream RNA processing such as splicing and 3’ end cleavage require a correctly capped transcript

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20
Q

How is the CTD involved in RNA capping?

A

High levels of Ser5P at the transcription start site (TSS) are needed for rapid RNA capping.
Ser5P and capping complex lead to a change in conformation which assists activation. In fission yeast,the phenotype for Ser5 substitution to Ala is lethal- highlights importance of Ser5 for capping.

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21
Q

Is Co-transcriptional splicing also regulated by CTD?How?

A

Yes, it is linked to Ser2. Mutations in Ser2 lead to a decrease in spliceosome recruitment showing importance.

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22
Q

Is the CTD involved in Transcription termination?

A

Yes

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23
Q

What is the Pre-Initiation Complex?

A

The set of proteins in addition to RNAPII that need to be recruited to start point of transcription in order to transcribe a gene

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24
Q

What general transcription factors or are required to direct accurate initiation of transcription?

A

TFIIA, TFIIB, TFIID,TFIIE,TFIIF and TFIIH

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25
What is basal transcription?
In vitro on naked DNA, the GTFs and RNAPII will specifically bind to a core promoter sequence and drive low levels of transcription.
26
Explain the stepwise model of assembly of the PIC
Initially TFIID binds the core promoter. TFIID is a big multi protein complex and contains a protein called TBP (TATA binding protein) which causes DNA to bend. TBP induced bending relieves repression between TBP and TAF11/13. Bending of DNA leads to recruitment of TFIIA. Then TFIIB binds. TFIIF binds RNAPII in solutions and brings it to the complex (RNAP is recruited in unphosphorylated form) TFIIF and RNAPII do not make specific interaction with the DNA . TFIIE binds and then TFIIH follows. TFIIH phosphorylates Ser5 of the CTD allowing transcription initiation to occur. Another protein within TFIIH ops up double stranded DNA forming transcription bubble = TRANSCRIPTION INITIATION.
27
Explain the Holoenzyme model
Genetic studies suggest that in a real cell there would be a pre assembled transcription complex. TFIID would first bind to core promoter. The remaining GTFS and RNAPII, including mediator and Co activators, would be recruited to the promoter as a preassembled (holoenzyme complex).
28
What is the TFIID complex comprised of?
TATA binding protein (TBP) 13-14 TBP Associated Factors (TAFs)
29
Can TAFs recognise modified histones?
Yes
30
What does a TBP-SAGA complex replace at some promoters?
TFIID
31
What does TFIIA do and how?
Stabilise the TBP-TATA complex by inhibiting TBP dimer formation and non productive interaction between TAF1 and TBP
32
What does TFIIB do?
Stabilise the TFIID/ promoter complex Recruites RNAPII/TFIIF by forming a bridge between TFIID and the RNAPII/TFIIF complex. Makes sequence specific contacts with the BRE
33
What two things are responsible for start site selection and how?
TFIIB and RNAPII. The structure (zinc finger) which inserts itself into RNAPII and part of TFIIB interacts with active site where RNA synthesis occurs and that’s responsible for start site selection
34
How long does TFIIF stay bound to RNAPII?
Until end of elongation
35
What are TFIIFs roles:
Facilitates recruitment of RNAPII to TFIID TFIIB promoter complex and stabilises it. Helps to recruit TFIIE and H Involved in start site selection and promoter escape
36
How is TFIIF involved in promoter escape?
It allows RNAPII to escape core promoter and enter elongation phase - suppresses pausing
37
What does TFIIE do?
Recruits TFIIH and stimulates the ATPase, CTD kinase and helicase activities of TFIIH
38
Describe TFIIH
Largest and most complex GTF. Contains CDK7 which is responsible for phosphorylation of SER5 within CTD prior to promoter escape. Contains an ATPase involved in transcription initiation Contains a helicase which opens DNA and induces promoter melting giving single stranded template. TFIIH is also involved in transcription coupled nucleotide excision repair.
39
What is Mediator?
A basal transcription factor, required in vivo for transcription of all pol II genes
40
Describe transcription initiation + Elongation:
Following PIC assembly, the complex undergoes conformational change. DNA is melted and template strand enters RNAPII active site. Transcription initiates. Short abortive products ar e made before a stable elongation complex is formed. Promoter contacts released once nascent RNA is approx 30 bases long. Elongation factors compete with GTFs to bind to RNAPII and facilitate promoter release.
41
What are the major DNA sequence elements involved in transcriptional control?
Core promoter- binds GTFs and RNAPII PPE- proximal promoter elements and enhancers- bind activator and repressor proteins
42
What are the three types of core promoters ?
Focused, dispersed or mixed
43
What is a focused corepromoter ?
When transcription starts at a defined point ( 1/2bp from that point)
44
What’s a dispersed promoter?
RNAPII starts at a no. Of positions usually over 100bp.
45
In humans which promoter is more common? And why?
Dispersed- vast majority of promoters in humans are dispersed. They are typically constitutively expressed and tend to drive transcription for housekeeping genes
46
A focused core promoter may contain one or more of what sequences?
BRE upstream,BRE downstream, MTE, DPE, TATA, DCE1, DCE2, DCE3
47
Most focussed promoters contain what?
TATA box and Inr element
48
What marks do focussed promoters have when active? Examples?
Specific Epigenetic marks. E.g. histones tend to be tri methylated at lysine 4 of histone 3 H3K4 me3 And lysine 27 of histone 3 is Acetylated H3 K27 ac
49
Characteristic of Dispersed promoters?
CpG islands
50
What are CpG islands?
Regions of DNA relatively rich in unmethylated CpGdinucleotides
51
Describe dispersed promoter for housekeeping genes
Flanked by precisely positioned nucleosomes Contains a single CpG island Generally constitutive Nucleosomes contain histone proteins which dill contain Epigenetic marks
52
Describe dispersed promoter for developmental transcription factors
Nucleosome precisely positioned Made up of long overlapping CpG islands of multiple CpG islands (diff between this and housekeeping) Regulated by Epigenetic factors
53
What are the mechanisms by which TFIID recognises the core promoter?
TFIID chromatin contacts TFIID DNA contacts (Co) activator TFIID contacts
54
Describe the recent proposal by Chen et al for a new mechanism of PIC formation
TFIID and TFIIA bind the core promoter TFIID, TFIIA, promoter DNA + TFIIB, PolII and TFIIF = cPIC (core pic) + TFIIE = mPIC (intermediate PIC) +TFIIH= hPIC (holo PIC)
55
What are the two tracks to formation of complete PIC following formation of TFIIA TFIID DNA complex ?
TATA-DPE (downstream promoter element) - for promoters with a TATA box plus Inr and DPE/MTE TATA only or TATA-less - only one major attachment point for TFIID
56
How many lobes does TFIID have ?
3, Lobe 1, 2 and 3
57
What does initial TFIID / TFIIA binding result in?
Results in TBP and love C of TFIID being separated by about 32 base pairs
58
What happens when TFIIB, TFIIF, RNAPII bind?
TBP and Lobe C move apart-
59
How do TBP and lobe c move apart in TATA only promoters?
Lobe C moves downstream
60
How do TBP and Lobe C move apart in TATA less promoters
TBP moves upstream
61
Explain the direct promoter deposition model
In TATA less and TATA only promoters When TFIIB, TFIIF and POLII bind there is reorganisation of the complex to position DNA close to PolII catalytic centre in the cPIC (drive conformation) - maintained in m and hPIC
62
Explain the stepwise promoter deposition model
TFIID makes strong contacts with different promoter elements - prohibits drive conformation when PolII binds. TFIIB, TFIIF and polII bind- park conformation (cPIC) TFIIE binds- significant change in complex- neutral conformation (mPIC) TFIIH binds- drive conformation (DNA in correct position relative to catalytic site of POLII) (hPIC)
63
What are alternative GTFs?
Tissue if gene specific GTFs e.g. TBP related factor TRFs can replace TBP. Tissue or gene specific basal transcription factors drive transcription of a subset of genes
64
How many copies of the histone genes are there?
Around 100
65
Is H1 expressed at same or different levels to H2A, H2B, H3 and H4?
Different
66
What does H1 utilise to drive transcription (of Histone 1)?
TRF2
67
What is Myogenesis?
Formation of skeletal muscular tissue (particularly during embryonic development)
68
In GTF recycling, what factors remain bound to DNA in a scaffold complex?
TFIID, TFIIA, mediator, TFIIE and TFIIH
69
Does chromatin environment act as a general activator or repressor.
Repressor
70
What other proteins are required in transcription besides GTFs and RNAPII
Activators!! Most genes require 2-3 activators and 1 repressor protein
71
What do Co activators do?
Form a bridge between activators and GTFs/RNAPII Modify or remodel chromatin to facilitate transcription
72
In addition to core promoter what other elements are required to enable activators to bind to DNA?
Proximal Promoter Element (PPE)- immediately upstream from core promoter Enhancer region ( up to 100kb up or downstream)
73
Transcription occurs in bursts, what determines the burst size and what determines the frequency?
Core promoter determines the size and enhancer determines the frequency
74
Explain modular enhancers
The precise position or order of diff activator binding sites is NOT important - as long as they bind
75
Explain non- modular enhancers :
Precise arrangement if activator binding sites is important and essential for enhancer function
76
Give an example for modular enhancer
Hunchback gene- binding sites for HB and BCD can be moved relative to each other
77
Give an example for non modular enhancers
Interferon Beta promoter
78
Describe regulation of TNF alpha gene
The TNF alpha gene enhancer binds different factors depending on stimuli- switched in by either increase in calcium or virus. Different activators bind to enhancer depending on the stimuli- but drive the same response.
79
How do most repressors work?
Either recruiting Co-depressors that put down negative epigenetic marks Or repressors that work to block activator at a particular Gene
80
What is passive repression?
The blocking of the action of an activator ( steric hindrance, masking, interacting with GTF, sequestering activator in cytoplasm)
81
What’s active repression?
Active repression generally works via chromatin structure ( epigenetic tags etc)
82
What is steric hindrance ?
Where the repressor binds a DNA site that overlaps an activator site
83
What is masking?
When the repressor binds the activator and masks the activation domain- the repressor may or may not bind to the DNA

Eukaryotic genetics (1 decks)

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