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Flashcards in Transcription Deck (80):

What is the main difference between transcription in prokaryotes and eukaryotes

In prokaryotes transcription/translation is instant.
In eukaryotes mRNA needs to travel out of the nucleus to be translated


What is a transcription 'bubble'

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


What are the three main steps in transcription



What is the structure of e.coli RNA polymerase

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)


Give an example of a conserved RNA polymerase binding site sequence

Thermus aquaticus:
Forms an aspartic acid loop - highly conserved


What is the importance of a protein bridge in RNA polymerase

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


What is a holoenzyme complex and why is it important

Holo = core enzyme + sigma factor
Core enzyme catalyses transcription; but sigma factor is needed for initiation.


How does sigma factor affect transcription initiation

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


How does a holoenzyme find/bind to a promoter sequence

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.


What is the difference between an open and closed DNA complex

Closed - DNA is double stranded (normal DNA structure)
Open - DNA is separated; transcription bubble has formed.


What is abortive initiation

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.


Where is the transcription 'bubble' in relation the strand

Between ~ -10 and +1 bps.


How many different sigma factors are present in e.coli

6/7 - 6 unique factors.


What is the most common sigma factor in e.coli

sigma70; coded by rpoD. Used for general transcription.


What are the three components of a promoter consensus sequence

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


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

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


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

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


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

Affects initial binding of RNA polymerase
(decreased - down-mutation; increased - up-mutation)


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

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


How can mutations in the consensus sequence be counteracted

Compensatory mutations in sigma factor.


What does 'K2' refer to

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


What effect does negative supercoiling have on transcription

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


Where does supercoiling occur during transcription

Positive supercoiling (ahead of RNA pol)
Negative supercoiling (behind RNA pol)


How is supercoiling (caused by transcription) rectified

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


Define positive and negative supercoiling

Positive - DNA becomes more tightly wound
Negative - DNA becomes less tightly wound


How does sigma factor 70 initiate transcription

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


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.


How can transcription be repressed

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


How can transcription be activated

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


How does CAP help activate transcription

Interacts with an alpha subunit of the RNA pol holoenzyme.


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

Hairpin loop
Backtrack pausing


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


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.


What is Rho

A site-specific termination factor
46kDa hexameric RNA-dependent ATPase


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)


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.


What can anti-termination proteins enable

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


What elements of DNA control transcription

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


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.


What are enhancers/silencers

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


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.


What is a core promoter

The minimum portion of a promoter required to initiate transcription.


What is regulated gene expression determined by

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


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


What are the three main nuclear RNA polymerases present in eukaryotes

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


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

Basal transcription machinery (inc RNA pol)


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


What does GTF stand for

General (/basal) Transcription Factor


What are the GTFs involved in RNA pol II transcription



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

(All letters begin TFII-)


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.


What role do TAFs play in transcription initiation

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


What role does TFIIA have in transcription initiation

It stabilises TFIID at the promoter site


What role does TFIIB have in transcription initiation

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


What role does TFIIE have in transcription initiation

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


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.


What does UCE stand for

Upstream Control Element


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)


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)


What are the GTFs involved in RNA pol III transcription



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.


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)


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''.


What does NELF stand for

Negative ELongation Factor


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


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)


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

P-TEFb (Positive Transcription Elongation Factor b)


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


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


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.


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)


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


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.


What is an enhanceosome

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


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)


How do activators function to stimulate transcription

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


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


What does PIC stand for

PreInitiation Complex


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.


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)