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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
Define positive and negative supercoiling
Positive - DNA becomes more tightly wound | Negative - DNA becomes less tightly wound
26
How does sigma factor 70 initiate transcription
Immediately active after translation, when bound to DNA and the core RNA pol
27
How does sigma factor 54 initiate transcription
Requires an ATP-dependent activation event (provided by a AAA+ ATPase) before can melt DNA and initiate transcription.
28
How can transcription be repressed
By a repressor protein. Binds to operator and blocks the promoter. Example; Lac repressor
29
How can transcription be activated
Activator binds upstream of promoter; activates transcription initiation. Example; CAP, FNR, I repressor
30
How does CAP help activate transcription
Interacts with an alpha subunit of the RNA pol holoenzyme.
31
What are the two types of pauses in the elongation phase of transcription
Hairpin loop | Backtrack pausing
32
How was the elongation process studied
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
33
How is transcription terminated by the process of intrinsic termination
Hairpin formed by G-C rich region. Followed by single stranded U-run (weakest bond). RNA pol just falls off - transcription terminates.
34
What is Rho
A site-specific termination factor | 46kDa hexameric RNA-dependent ATPase
35
How is transcription terminated by Rho
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
How can transcription termination be prevented
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
What can anti-termination proteins enable
``` Multicistronic transcription (more than 1 gene per transcript) Co-regulation (in operons). ```
38
What elements of DNA control transcription
Core promoter and promoter proximal sequences (close to start site) Enhances/silencer sequences (from a distance)
39
How are promoters defined
Defined by their ability to cause transcription. | Tested by removed nucleotides from the test promoter, and seeing how many are required.
40
What are enhancers/silencers
Sequence that is able to module (up or down) the levels of initiation at the promoter region
41
How do enhancers modulate promoter regions from a distance
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
What is a core promoter
The minimum portion of a promoter required to initiate transcription.
43
What is regulated gene expression determined by
``` Promoter + enhancers Transcription factors (signal responsive/cell type specific/constitutive) ```
44
How can long distance transcription controllers be regulated
Insulators/boundary elements (BE) - Blocks enhances from other genes getting involved Matrix Attachment regions (MAR) - Attaches to nuclear matrix, creates chromosomal domains
45
What are the three main nuclear RNA polymerases present in eukaryotes
RNA pol 1 (rRNAs) RNA pol 2 (mRNAs) RNA pol 3 (tRNAs/snRNAs)
46
What are the 3 classes of factor needed for regulated transcription initiation
Basal transcription machinery (inc RNA pol) Activators Co-activators
47
What are the roles of activators and co-activators in transcription initiation
Activators bind at promoter, and to other distal sites (promoter/enhancer) Coactivators connect activators to the basal factors
48
What does GTF stand for
General (/basal) Transcription Factor
49
What are the GTFs involved in RNA pol II transcription
TFII- A-H
50
In what order do the GTFs involved in RNA pol 2 assemble
(All letters begin TFII-) | DAB[Pol2]FEH
51
What precedes the assembly of transcription factors on a class 2 core promoter
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
What role do TAFs play in transcription initiation
Contribute to promoter strength/selectivity. Along with TBP allows TFIID to bind.
53
What role does TFIIA have in transcription initiation
It stabilises TFIID at the promoter site
54
What role does TFIIB have in transcription initiation
Bridges TFIID and Pol II. Determines the transcription start site.
55
What role does TFIIE have in transcription initiation
Involved in promoter opening (DNA unwinding) and regulating TFIIH activity
56
What role does TFIIH have in transcription initiation
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
What does UCE stand for
Upstream Control Element
58
What are the GTFs involved in RNA pol 1 transcription
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
What are the two types of promoters recognised by RNA pol 3
``` Internal promoters (downstream (further along) of start point - 5S and tRNA) Upstream promoters (upstream (behind) of start point - snRNAs) ```
60
What are the GTFs involved in RNA pol III transcription
TFIII- A/B/C
61
In what order do the GTFs involved in RNA pol 3 assemble
TFIII-A/C bind. Recruit TFIIIB to the start point. TFIIIB recruits RNA pol III. Transcription can occur.
62
What is the role of TFIIIA and TFIIIC in transcription initiation
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
What is the role of TFIIIB in transcription initiation
Positioning factor. Recruits RNA Pol III. TFIIIB is a complex consisting of TBP, BRF and B''.
64
What does NELF stand for
Negative ELongation Factor
65
What is the role of elongation factors in eukaryotic transcription
Increase the rate of elongation Assist Pol II through pause sites Facilitate transcription through chromatin Provide a platform for chromatin remodelling activities
66
What are the main elongation factors involved in increasing the rate of elongation
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
What are the main elongation factors involved in assisting RNA pol II through transient pause sites
TFIIS. | P-TEFb (Positive Transcription Elongation Factor b)
68
How does TFIIS assist RNA pol II through transient pause sites
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
How does P-TEFb assist RNA pol II through transient pause sites
Displaces NELF, and modifies RNA pol II, releasing it from the pause site
70
What are the main elongation factors involved in assisting RNA pol II transcribe through chromatin
FACT (FAcilitates Chromatin Transcription) - Acts as a histone chaperone and can displace a H2A-H2B dimer from a nucleosome.
71
What are the main elongation factors involved in elongating RNA pol II
PAF1 complex (Polymerase Associated Factor 1) - A platform for histone modifying activity (deals with nucleosomes)
72
How do transcriptional activators regulate gene expression
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
What are common types of DNA-binding domains for activators of transcription
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
What is an enhanceosome
A multitude of transcription factors that assemble into a macromolecular complex at enhancer sequences.
75
What is the role of an enhanceosome
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
How do activators function to stimulate transcription
Facilitate promoter opening (recruit chromatin remodellers) and recruitment/stabilisation of GTFs at core promoter (countering neg factors).
77
Describe mechanisms by which transcription activators might function
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
What does PIC stand for
PreInitiation Complex
79
How do co-activators function to facilitate transcription
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
How can transcriptional activator functions be regulated
* 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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