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3014 > Eukaryotic RNAP II Transcription Machinery and CTD Regulation > Flashcards

Eukaryotic RNAP II Transcription Machinery and CTD Regulation Flashcards

1
Q

What are the three different RNAP enzymes found in animals and what are their roles?

A
  • RNApoly I – Most rRNA
  • RNApoly II – All protein coding genes
  • RNApoly III – 5S rRNA, tRNA, small nuclear RNA
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2
Q

What are operators an example of?

A

Proximal regulatory sequences

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3
Q

How far away from the TSS are core promoters?

A

±50bp

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4
Q

How far away from the TSS are Proximal regulatory sequences?

A

50-1000bp, as these often curl around to make contact with the PIC

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5
Q

What is a core promoter?

A

the minimal unit required for transcription

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6
Q

What are the two classes of core promoter?

A

TATA-box and TATA-less promoters.

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7
Q

What are TATA-less promoters used for?

A

be housekeeping genes that require less careful regulation. Very often a TATA-less promoter will be a broad promoter, which is one with multiple transcription start sites. Transcription at these promoters is poorly understood.

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8
Q

What are TATA-box promoters?

A

They are very well studied despite being present in only around a quarter of human genes. These are used in the more tightly regulated genes and hence are better studied.

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9
Q

Where is the TATA box relative to the TSS, and why?

A

The TATA box is always at -30bp, as the TATA box is used to define the TSS

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10
Q

What other elements can a TATA-box include and what do these do?

A

a TFIIB recognition element (BRE), an initiator element (Inr) and a downstream promoter element (DPE), all of which bind different transcription factors.

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11
Q

What is the Pre-Initiator Complex (PIC)?

A

the colossal complex of transcription factors that recruit the Pol II itself

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12
Q

What is the advantage to having a large number of recruitment steps for the PIC?

A

GTFs build up in a stepwise way, each addition having the ability to be regulated. This means that the large number of steps leads to a high sensitivity to regulation.

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13
Q

Which is the first GTF to bind?

A

TFIID (transcription factor for RNApoly II D).

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14
Q

What is TFIID?

A

a huge complex of proteins made up of the TATA-box Binding Protein (TBP) and 14-18 TBP Associated Factors (TAFs)

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15
Q

How conserved is TBP?

A

TBP is evolutionarily conserved across all of life, even archaea

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16
Q

Why is TBP so conserved?

A

Because it binds and reshapes the TATA-box this step determines the TSS for the transcription and acts as a nucleation point for all GTFs

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17
Q

What are the second and third TFs to bind into the PIC?

A

TFIIB and A

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18
Q

What does TFIIA do?

A

stabilises the TFIID-DNA complex

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19
Q

What does TFIIB do?

A

further helps TSS determination/TATA reshaping and stabilises the initial opening of the DNA. TFIIB directly interacts with the Pol II

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20
Q

What is the fourth TF to be recruited to the PIC, after B and A?

A

TFIIF, which is bound to the Pol II. At this step the main enzyme is recruited and the complex is rearranged.

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21
Q

What is the 5th TF to bind into the PIC after F/PolII recreuitment, and what does it do?

A

TFIIE, whose main purpose seems to be just to recruit the last transcription factor TFIIH, but is also thought to stabilise the non-template DNA strand.

22
Q

What is the 6th and final TF to be recruited to the PIC, and what does it do?

A

TFIIH is a large complex that has both 3’-5’ and 3’-5’ helicase activity.
This is also a kinase that phosphorylates Ser5 in the Pol II (CTD), which is key to forming an open complex and for promoter clearance.

23
Q

How much transcription will occur when the PIC is present?

A

for only a very low baseline transcriptional rate, in order to produce considerable amounts of any protein there must be enhancers, transcription factors and other upregulatory factors in play.
Hence it is also called the basal transcription complex, as it is the MINUMUM required for transcription.

24
Q

What are the four stages of Pol II GTF assembly?

A
  1. TBP/TFIID recruitment to the TATA-box
  2. TFIIA/TFIIB recruitment
  3. Pol II/TFIIF recruitment
  4. TFIIE/TFIIH recruitment
25
Q

What is the most important activator of the PIC?

A

The mediator complex. This is a huge complex whose components act as general coactivators for Pol II and several of the GTFs

26
Q

How does the mediator complex work?

A

By binding over the entire PIC, directly interacting with the Pol II and the GTFs, stabilising the PIC assembly and increasing its activity.

27
Q

What is promoter clearance?

A

This is the term used to refer to the Pol II escaping from the PIC and beginning transcription.

28
Q

What must happen in order for the RNA Polymerase II to produce an mRNA transcript? What TF causes this?

A

the DNA must first be opened at the promoter and loaded into the RNAP by TFIIH

29
Q

How is the DNA opened at the promoter during initiation?

A

The DNA is unwound and pushed into a bubble by the 3’-5’ helicase domain XPB of TFIIH when the DNA is pushed into the Pol II cavity.

30
Q

What part of TFIIH performs the CTD phosphorylation?

A

CDK7 phosphorylates Ser-5 (Kin28 dependent phosphorylation)

31
Q

What active subunits does TFIIH have and where does it bind DNA?

A

the 3’-5’ helicase domain XPB
a 5’-3’ helicase domain called XPD
CDK7 kinase region
TFIIH binds to the DNA in two places, one of which is near to the CTD.

32
Q

What happens after the TFIIH phosphorylation?

A

After the Kin28 dependent phosphorylation the mediator dissociates, which allows Pol II to escape the PIC and begin transcription.

33
Q

What is left behind after promoter clearance, and why?

A

The post-escape complex of the GTFs (minus the mediator and TFIIF, which proceeds with Pol II).

When the gene is being upregulated the mediator and a new Pol II are recruited to the complex without needing to re-assemble the entire PIC.

34
Q

What is the role of the CTD, and how does it function?

A

The carboxy terminal domain is a regulatory hub that co-ordinates all the different steps of transcription (initiation, elongation and termination) and pre-mRNA processing (capping, splicing and nuclear export, 3’ cleavage and polyadenylation).
This is primarily accomplished through phosphorylation of various residues.

35
Q

What is the structure of the CTD?

A

A long tail on the Pol II consisting of many repeats of a conserved seven residue sequence, a heptapeptide of sequence YSPTSPS.
Note the high number of phosphorylatable tyrosine, serine and threonine residues.

36
Q

How does Pol II CTD structure differ in different species?

A

Pol II in different species differ in the number of repeats of the sequence, yeast have 26, mice 44 and humans 52.

Human CTDs also have some sequence variations not seen in yeast or mice.

37
Q

Where can the CTD repeat be phosphorylated, and which are most important?

A

Phosphorylation on the heptapeptide can occur at Y1, S2, T4, S5 and S7.

The phosphorylations at S2 and S5 are the most important.

38
Q

What is the role of S2 and S5 phosphorylation during transcription?

A

S5 phosphorylation stimulates promoter escape and S2 phosphorylation causes termination.

Both phosphorylations upregulate elongation.

39
Q

What is the role of S2 and S5 phosphorylation during pre-mRNA processing?

A

During pre-mRNA processing S5 phosphorylation stimulates capping, S2 phosphorylation causes 3’ end cleavage and polyadenylation.

Both upregulate splicing and nuclear export.

40
Q

What happens to the relative levels of S2 and S5 phosphorylation during transcription?

A

The relative levels of S2 and S5 phosphorylation invert, S5 starting high and falling as S2 rises.

This relates to the promoter escape and termination stages, with a fairly constant total amount of S2 + S5 phosphorylation constant to continue elongation.

41
Q

Other than transcription and pre-mRNA processing, what else does the CTD regulate?

A

All the possible phosphorylations also have an impact of post-transcriptional modification.

42
Q

What regulates the CTD?

A

All of the phosphorylations are regulated by a whole set of kinases and phosphatases.

43
Q

What is the role of S7 phosphorylation?

A

The role of S7 phosphorylation is nor clearly understood, but it is almost constantly phosphorylated.

44
Q

Other than phosphorylation, how is the CTD regulated?

A

cis-trans isomerisation of the prolines in the YSPTSPS

45
Q

How complex is the CTD code?

A

Incredibly so, it is thought that regulatory proteins can read the phosphorylation levels and the proline cis-trans isomerisation meaning that every residue in the heptapeptide can be changed.

46
Q

What catalyses the interconversion between the proline conformations?

A

a proline isomerase called ESS1/PIN1.

47
Q

how many possible variations of CTD structure are there in humand and in yeast?

A

64^52 in humans and 64^25 in Saccharomyces cerevisiae.

48
Q

What is PCF11?

A

PCFII is a protein that binds to the heptapeptide sequence by its CTD Interacting Domain (CID).

49
Q

How does PCF11 bind to the CTD?

A

Rather than just recognising a single repeat PCF11 actually binds across one whole sequence and extends back through to the S5 of the previous repeat.

50
Q

What dictates the PCF11 affinity for the CTD?

A

PCF11 will only bind when it is stabilised by the phosphorylation of S2. It also requires the two prolines in the main motif bound to both be in the trans configuration.

The affinity of PCF11 also increases when the adjacent sequences are also in the right conformation.

3014 (14 decks)

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