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Transcription Flashcards

1
Q

What is the main difference between transcription in prokaryotes and eukaryotes

A

In prokaryotes transcription/translation is instant.

In eukaryotes mRNA needs to travel out of the nucleus to be translated

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2
Q

What is a transcription ‘bubble’

A

The place where the two strands of DNA are separate and being transcribed.
Bubble length ~12-14 bp

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3
Q

What are the three main steps in transcription

A

(Pre-initiation)
Initiation
Elongation
Termination

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4
Q

What is the structure of e.coli RNA polymerase

A

Core enzyme made up of two parts:
Alpha 1+2 subunits dimerise and bind to beta subunit (alpha2beta)
Beta’ subunit binds to omega subunit (beta’omega)
These two subunits combine to form the core enzyme (alpha2betabeta1omega)

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5
Q

Give an example of a conserved RNA polymerase binding site sequence

A

Thermus aquaticus:
-NADFDGD-
Forms an aspartic acid loop - highly conserved

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6
Q

What is the importance of a protein bridge in RNA polymerase

A

Changes the conformation. Restricts nucleotide entry - controls/regulates transcription.

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7
Q

What is a holoenzyme complex and why is it important

A

Holo = core enzyme + sigma factor

Core enzyme catalyses transcription; but sigma factor is needed for initiation.

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8
Q

How does sigma factor affect transcription initiation

A

Binds to ‘core’ polymerase enzyme. It reduces affinity to non-specific DNA; and increases affinity for promoters.

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9
Q

How does a holoenzyme find/bind to a promoter sequence

A

Rapidly binds to random DNA.
‘Slides’ or ‘hops’ along DNA until it finds a promoter - forms a tight complex.
1D diffusion - limits how far it can go.

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10
Q

What is the difference between an open and closed DNA complex

A

Closed - DNA is double stranded (normal DNA structure)

Open - DNA is separated; transcription bubble has formed.

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11
Q

What is abortive initiation

A

Lots of short sequences (<8 bp) are formed. Sigma factor is released when sequence reaches 8-9 bp; so transcription is stopped before then if sequence is not required.

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12
Q

Where is the transcription ‘bubble’ in relation the strand

A

Between ~ -10 and +1 bps.

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13
Q

How many different sigma factors are present in e.coli

A

6/7 - 6 unique factors.

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14
Q

What is the most common sigma factor in e.coli

A

sigma70; coded by rpoD. Used for general transcription.

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15
Q

What are the three components of a promoter consensus sequence

A

hexonucleotide at ~ -35
hexonucleotide at ~ -10 (TATA box)
Start point.

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16
Q

What sequences does sigma factor (70) recognise and direct RNA polymerase to.

A 
Two hexonucleotides (6 bp sequence)
One at ~-35; then TATA box at ~-10
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17
Q

What effect do down-mutations of consensus sequence have on promoter efficiency

A

Decreased efficiency.
Usually because decreased conformance to consensus sequence.
(Up-mutations do the opposite)

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18
Q

What specific effect do mutations in the -35 consensus sequence have

A

Affects initial binding of RNA polymerase

decreased - down-mutation; increased - up-mutation

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19
Q

What specific effect do mutations in the -10 consensus sequence have

A

Affects the melting reaction that converts a closed complex to an open complex.
(decreased - down-mutation; increased - up-mutation)

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20
Q

How can mutations in the consensus sequence be counteracted

A

Compensatory mutations in sigma factor.

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21
Q

What does ‘K2’ refer to

A

The measure of the activity of a promoter (promoter strength)

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22
Q

What effect does negative supercoiling have on transcription

A

Increases efficiency of certain promoters by assisting the melting reaction (bubble formation).

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23
Q

Where does supercoiling occur during transcription

A 
Positive supercoiling (ahead of RNA pol)
Negative supercoiling (behind RNA pol)
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24
Q

How is supercoiling (caused by transcription) rectified

A

Positive supercoiling - Gyrase introduces negative supercoils
Negative supercoiling - Topoisomerase relaxes negative supercoils

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25
Q

Define positive and negative supercoiling

A

Positive - DNA becomes more tightly wound

Negative - DNA becomes less tightly wound

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26
Q

How does sigma factor 70 initiate transcription

A

Immediately active after translation, when bound to DNA and the core RNA pol

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27
Q

How does sigma factor 54 initiate transcription

A

Requires an ATP-dependent activation event (provided by a AAA+ ATPase) before can melt DNA and initiate transcription.

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28
Q

How can transcription be repressed

A

By a repressor protein. Binds to operator and blocks the promoter.
Example; Lac repressor

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29
Q

How can transcription be activated

A

Activator binds upstream of promoter; activates transcription initiation.
Example; CAP, FNR, I repressor

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30
Q

How does CAP help activate transcription

A

Interacts with an alpha subunit of the RNA pol holoenzyme.

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31
Q

What are the two types of pauses in the elongation phase of transcription

A

Hairpin loop

Backtrack pausing

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32
Q

How was the elongation process studied

A

Stuck a single RNA pol onto a bead.
RNA pol stationary, so the strand moved. Measured rate of movement/nucleotide used up. Calculated velocity (bp/s). Various speeds observed

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33
Q

How is transcription terminated by the process of intrinsic termination

A

Hairpin formed by G-C rich region.
Followed by single stranded U-run (weakest bond).
RNA pol just falls off - transcription terminates.

34
Q

What is Rho

A

A site-specific termination factor

46kDa hexameric RNA-dependent ATPase

35
Q

How is transcription terminated by Rho

A

Rho binds to a ‘rut’ site (C-rich) in the transcribed RNA.
Rho slides up the RNA and catches up with RNA pol (when paused at a terminator sequence)
Rho unwinds the RNA/DNA hybrid structure (transcription bubble) causing release of all factors (DNA goes back to normal)

36
Q

How can transcription termination be prevented

A

Anti-termination proteins can act on RNA pol and allow it to bypass a terminator region.
Anti-terminators act upstream of the terminator site.

37
Q

What can anti-termination proteins enable

A 
Multicistronic transcription (more than 1 gene per transcript)
Co-regulation (in operons).
38
Q

What elements of DNA control transcription

A

Core promoter and promoter proximal sequences (close to start site)
Enhances/silencer sequences (from a distance)

39
Q

How are promoters defined

A

Defined by their ability to cause transcription.

Tested by removed nucleotides from the test promoter, and seeing how many are required.

40
Q

What are enhancers/silencers

A

Sequence that is able to module (up or down) the levels of initiation at the promoter region

41
Q

How do enhancers modulate promoter regions from a distance

A

DNA is flexible. It is thought that DNA looping can occur, bringing the enhancer regions close to the RNA pol binding site; enhancing transcription.

42
Q

What is a core promoter

A

The minimum portion of a promoter required to initiate transcription.

43
Q

What is regulated gene expression determined by

A 
Promoter + enhancers
Transcription factors (signal responsive/cell type specific/constitutive)
44
Q

How can long distance transcription controllers be regulated

A

Insulators/boundary elements (BE) - Blocks enhances from other genes getting involved
Matrix Attachment regions (MAR) - Attaches to nuclear matrix, creates chromosomal domains

45
Q

What are the three main nuclear RNA polymerases present in eukaryotes

A

RNA pol 1 (rRNAs)
RNA pol 2 (mRNAs)
RNA pol 3 (tRNAs/snRNAs)

46
Q

What are the 3 classes of factor needed for regulated transcription initiation

A

Basal transcription machinery (inc RNA pol)
Activators
Co-activators

47
Q

What are the roles of activators and co-activators in transcription initiation

A

Activators bind at promoter, and to other distal sites (promoter/enhancer)
Coactivators connect activators to the basal factors

48
Q

What does GTF stand for

A

General (/basal) Transcription Factor

49
Q

What are the GTFs involved in RNA pol II transcription

A

TFII- A-H

50
Q

In what order do the GTFs involved in RNA pol 2 assemble

A

(All letters begin TFII-)

DAB[Pol2]FEH

51
Q

What precedes the assembly of transcription factors on a class 2 core promoter

A

TBP (TATA binding protein)(in a TAF complex(TBP-Associated Factors)) binds to the TATA box through DNA minor groove recognition. This allows the first transcription factor (TFIID) to bind.

52
Q

What role do TAFs play in transcription initiation

A

Contribute to promoter strength/selectivity. Along with TBP allows TFIID to bind.

53
Q

What role does TFIIA have in transcription initiation

A

It stabilises TFIID at the promoter site

54
Q

What role does TFIIB have in transcription initiation

A

Bridges TFIID and Pol II. Determines the transcription start site.

55
Q

What role does TFIIE have in transcription initiation

A

Involved in promoter opening (DNA unwinding) and regulating TFIIH activity

56
Q

What role does TFIIH have in transcription initiation

A

Phosphorylates the CTD (C-terminal domain) of Pol II. This triggers release of Pol 2 (promoter clearance)
Also part of a ‘repair complex’ during elongation stage.

57
Q

What does UCE stand for

A

Upstream Control Element

58
Q

What are the GTFs involved in RNA pol 1 transcription

A

SL1 (TBP/TAF complex) - binds to the core promoter; stabilises UBF and recruits RNA pol 1
UBF (architectural protein; bends DNA) - activates transcription (through anti-repression and stimulating promoter clearance)

59
Q

What are the two types of promoters recognised by RNA pol 3

A 
Internal promoters (downstream (further along) of start point - 5S and tRNA)
Upstream promoters (upstream (behind) of start point - snRNAs)
60
Q

What are the GTFs involved in RNA pol III transcription

A

TFIII- A/B/C

61
Q

In what order do the GTFs involved in RNA pol 3 assemble

A

TFIII-A/C bind. Recruit TFIIIB to the start point. TFIIIB recruits RNA pol III. Transcription can occur.

62
Q

What is the role of TFIIIA and TFIIIC in transcription initiation

A

Assembly factors. Sole role is to recruit TFIIIB to the site
TFIIIA binds to Box A (5S-rRNA)
TFIIIC binds to Box C (5S-rRNA) and Box A/B (tRNA)

63
Q

What is the role of TFIIIB in transcription initiation

A

Positioning factor.
Recruits RNA Pol III.
TFIIIB is a complex consisting of TBP, BRF and B’’.

64
Q

What does NELF stand for

A

Negative ELongation Factor

65
Q

What is the role of elongation factors in eukaryotic transcription

A

Increase the rate of elongation
Assist Pol II through pause sites
Facilitate transcription through chromatin
Provide a platform for chromatin remodelling activities

66
Q

What are the main elongation factors involved in increasing the rate of elongation

A

ELLs (increase catalytic rate of pol II)
Elongin A/B/C (required for heat shock gene expression. Helps restarting pol II at pause sites on developmentally regulated genes)
DSIF (can affect positively or negatively)

67
Q

What are the main elongation factors involved in assisting RNA pol II through transient pause sites

A

TFIIS.

P-TEFb (Positive Transcription Elongation Factor b)

68
Q

How does TFIIS assist RNA pol II through transient pause sites

A

When RNA pol II pauses, continues to transcribe - in reverse (retrograde motion). Causes transcription arrest. TFIIS cleaves the extra transcribed bit, allowing RNA pol II to continue

69
Q

How does P-TEFb assist RNA pol II through transient pause sites

A

Displaces NELF, and modifies RNA pol II, releasing it from the pause site

70
Q

What are the main elongation factors involved in assisting RNA pol II transcribe through chromatin

A

FACT (FAcilitates Chromatin Transcription) - Acts as a histone chaperone and can displace a H2A-H2B dimer from a nucleosome.

71
Q

What are the main elongation factors involved in elongating RNA pol II

A

PAF1 complex (Polymerase Associated Factor 1) - A platform for histone modifying activity (deals with nucleosomes)

72
Q

How do transcriptional activators regulate gene expression

A

Bind to a sequence-specific DNA-binding domain.
Interact [in]directly with parts of the transcription apparatus.
DNA-binding/transcription-activating domains are separate; binding to the DNA brings the activator into the vicinity of the promoter

73
Q

What are common types of DNA-binding domains for activators of transcription

A

Zinc finger motif -
C terminal forms an alpha helix that binds to one turn of DNA; major groove. Usually multiple ‘fingers’ in a row; incorporates Zinc)
Leucine zippers - Amphipathic helix that dimerises. Dimerisation forms a bZIP motif - the two basic regions symmetrically bind to a palindromic DNA sequence.

74
Q

What is an enhanceosome

A

A multitude of transcription factors that assemble into a macromolecular complex at enhancer sequences.

75
Q

What is the role of an enhanceosome

A

To facilitate the assembly of the pre-initiation complex at the start site of transcription by interacting with:
GTFs (directly) and co-activators (that facilitate other stuff)

76
Q

How do activators function to stimulate transcription

A

Facilitate promoter opening (recruit chromatin remodellers) and recruitment/stabilisation of GTFs at core promoter (countering neg factors).

77
Q

Describe mechanisms by which transcription activators might function

A

Chromatin decondensation (through recruitment of remodelling complexes)
Promoting formation of the pre-initiation complex (by inducing conformational changes)
Recruiting TFIID to the promoter
Covalently modifying GTFs
Assisting with promoter clearance/elongation

78
Q

What does PIC stand for

A

PreInitiation Complex

79
Q

How do co-activators function to facilitate transcription

A

Facilitate promoter opening (by ATP-dependent chromatin remodelling and histone modifying complexes)
Act as a ‘mediator’ complex - forming a physical/functional bridge between activator and GTFs.

80
Q

How can transcriptional activator functions be regulated

A
  • Control TF production (gene expression; alt splicing; mRNA degradation; translation; feedback control)
  • Regulate TF localisation (keep unnecessary TFs out of the nucleus)
  • Regulate TF activity (e.g. modification that alter activity)
  • Regulate TF dimerisation (by altering concentrations)
  • Regulate TF proteolysis
  • Ligand binding (e.g. steroid hormones. Binding triggers release of complex which converts into a transcriptionally active form)

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