Transcription Flashcards
Types of RNA Polymerases: (Note: Type, not classes)
Phage Enzymes e.g. T7 RNA Pol. , single polypeptides
Bacterial Enzymes e.g. E.coli RNA Pol. multiple polypeptides
Eukaryotic Enzymes, multiple classes of RNA Pol. multiple polypeptides, plus additional factors
What is the general function of an RNA polymerase
RNA polymerase separates the two strands of DNA in a transient (short lasting/impermenant) “bubble” (Transcription bubble, about 12-14bp long), and uses the strand running from 3’ to 5’ to synthesise a complementary RNA sequence running from 5’ to 3’
DNA supercoils in transcription and how they can be dealt with.
Transcription can create superhelical tension in DNA strands, causing positive or negative supercoiling.
Supercoils formed when DNA has fixed ends and unwinds 10 base pairs (one helical turn)
Positive supercoiling occurs “ahead/downstream” of RNA pol. and hinders helix opening.
Negative supercoiling occurs “behind/upstream” RNA pol. and facilitates helix opening.
DNA topoisomerases can alter (‘reset’) the supercoiling of the DNA.
Class I DNA topoisomerases: Break 1 strand, turn, then re-ligate.
Class II DNA topoisomerases: Break both strand, and then religate.
General 8 step Overview of Transcription in Prokaryotes
- Closed complex forms at promoter (Sigma factor sliding)
- Open complex (Separated strands, Transcription bubble)
- Abortive Initiation (Abortive transcripts produced)
- Promoter Clearance and sigma factor release
- Elongation
- Termination hairpin formation (Causing RNA pol. to stall)
- Termination (polyU sequence causes dissociation of RNA pol.)
- Reassociation with sigma factor
What is Holo-Enzyme?
RNA polymerase + Sigma factor, binds to promoter in prokaryotic transcription.
Holo-enzyme binds very rapidly to random DNA, it finds the promoter via rapid exchange of bound sequences by “Sliding” (Hopping along until the promoter is found), it can then form a “tight/closed complex”
Transcription start signals
Signals that are encoded in DNA, telling RNA pol. where to start (also signals for stopping).
-10 and -35 consensus sequences allow binding of holo-enzyme through sigma factor
Note the -10 here is not a TATA box like in eukary, but is a TATAAT sequence, known as a Pribnow box
The closer to consensus the sequences are, the greater the binding, and so the greater the expression. This is known as “promoter strength”
Classes of RNA Polymerase (Note: Classes not Type)
Class I, II, and III
Class I: Synthesises rRNAs
Class II: Synthesises precursors of mRNAs and snRNAs, used in transcription initiation.
Class III: Synthesises tRNAs
General transcription factors (GTFs) of RNA pol. II
Required since RNA pol. cannot recognise a gene promoter by itself.
TFIID: TBP subunit - Recognises the TATA box
TAF subunits (11 of them) - Recognise other DNA
sequences near the transcription start point,
contribute to promoter selectivity and strength.
TFIIB: Recognises BRE (B recognition element, near the TATA box) in promoters, accurately positions RNA pol. at the start site of transcription.
TFIIF: Stabilises RNA pols interaction with TBP and TFIIB and helps attract TFIIE and TFIIH
TFIIE: Attracts and regulates TFIIH
TFIIH: Unwinds DNA. Phosphorylates the Ser5 residue of the RNA pols CTD (C-terminal domain), triggers promoter escape of RNA pol II (i.e. release of RNA pol. from the promoter). TFIIH completes the assembly of the PIC (pre-initiation complex). Helicase subunits have role in nucelotide excision repair during elongation
mRNA 5’ cap
Occurs in Elongation. Prevents degradation of mRNA, vital in the creation of stable and mature mRNA, promotes translation.
3’ polyadenylation tail
Occurs in Elongation. Prevents degradation, promotes translation.
Order of GTF (Pol.II) binding in transcription initiation in eukaryotes.
TFIID -> TFIIB -> TFIIF -> TFIIE + TFIIH
GTFs (Pol. III)
TFIIIC and TFIIIA: Assembly factors
TFIIIB: Complex of TBP, BRF, and B”. Essential for recruitment of Pol. III
GTFs (Pol. I)
SL1: TBP-TAF complex, binds the core promoter and stabilising UBF, essential for recruitment of Pol I
UBF: Activates transcription by anti-repression and promoter clearance
What regulates the activity of transcription?
A eukaryotic gene control region consists of a ‘core promoter’ plus many ‘cis-regulatory sequences’.
Trans-acting factors (Activators and repressors) bind to cis-regulatory sequences (Enhancers or Silencers)
They can be very far from the promoter, they modulate levels of initiation at the promoter.
Trans-acting factors
Modular proteins with multiple distinct domains.
Modulate levels of initiation.
The role of their DNA-binding domain is to bring their transcription-activation domain into the vicinity of the promoter.
