Lecture 4: Regulation of Transcription Initiation Flashcards
What are sigma factors (subunits) in RNA polymerase?
They are the specificity factor of RNA polymerase, binding to a specific promoter and open complex formation. Acts as an INITIATION FACTOR, accelerating the search for promoter DNA.
What do Sigma factors do?
- increase initiation rate (but not elongation rate)
- contacts with core occur throughout its length
- Cant bind to DNA unless bound to core
- Decreases core affinity to non-specific DNA
- Recognizes bases in coding strand at -10 and also double strands at -35
Is there more than one type of sigma factor?
Yes, various factors recognise different promoters and have different ‘regulons’
What is Sigma 54? Why is it unusual?
It is involved in activation of genes for nitrogen starvation conditions.
It is unusual because it can bind to the promoter in the absence of the Core RNA polymerase BECAUSE it has NO inhibitory domain (domain 1), therefore it is more like an eukaryotic transcription factor.
However, Sigma 54 and core RNA polym. can only facilitated closed complex formation, specific activators needed for open complex formation and successful transcriptional activation.
How does DNA and RNA polymerase interact?
- Core RNA polymerase is “stored” at nonspecific DNA sites before sigma binding.
- By reducing stability of the loose complexes, sigma allows the process to occur much faster, by stabilizing tight binding sites, open complexes occur irreversibly much faster
- When sigma is released, the core protein + nascent transcript is essentially locked onto the DNA until termination.
What is abortive initiation (or cycling)?
RNA pol. transcribe 2-9 bases then restarts. NEVER LEAVES THE PROMOTER.
This may occur several hundred times before elongation.
note; only 8 bases can fit in the active site, which correlates with the abortive products of 8-10 bases.
Explain the sigma cycle.
- closed promoter complex with sigma bound to -10 region (with RNA pol)
- open promoter complex formation, high promoter affinity due to sigma
- Sigma actor dissociates from core polymerase once a few elongation reactions have occurred
- Sigma can re-bind to other core enzymes
Elongation, how fast does it occur, and what happens?
- RNA pol transcribes at 30-50nt a second
- Rapid untwisting and re-annealing of DNA
- Part of the new RNA strand is hybrid DNA-RNA but most RNA is displaced as the helix reforms
How long (in turns of bases) is the transcription bubble of RNA polymerase? How long is the RNA/DNA hybrid?
Transcription bubble: 12-20 bases
RNA/DNA hybrid: 3bp
What DNA strand does mRNA look identical to?
The coding strand, EXCEPT from U in place of T
What two enzymes are required for management of super coiling? Where are they located and what do they do?
- Gyrase: located in front of the RNA polymerase and introduces negative super coils (loosening of double helix)
- Topisomerase: located behind RNA polymerase and positively super coils DNA (brings it back to normal resting state)
What are the two types of transcription termination? How do they occur?
- Factor independent: inverted repeats of GC followed by several T’s (+A’s) on the CODING strand - forms an mRNA hairpin loop which is very stable followed by the unstable UA region - RESULT; mRNA pulls from the bubble, therefore destabilizing the transcription bubble causing RNA polymerase to fall off
- Factor dependent: Proteins interact with polymerase or transcript to stop transcription - three termination factors in E. coli; Rho, Tau, NusA
How does Rho-dependent termination occur?
- only works when RNA isn’t being translated
- Rho is RNA-dependent ATPase; only cleaves ATP if RNA is present
- Rho is RNA-DNA helicase - will only unwind double helix if RNA:DNA hybrid
- if rut is being translated, termination cannot occur
- Rho binds to rut, sliding along, chasing polymerase - this requires ATP hydrolysis
- RNAP pauses at termination site - Rho catches polymerase
RHO ONLY BINDS TO MRNA
