L1: Prokaryotic Transcription Flashcards
At what levels can gene expression be regulated?
- During transcription
- During translation
- Post-transcriptional
Give the 3 types of functional RNA in the cell
- mRNA
- tRNA
- rRNA
RNA polymerase terminology:
- Core enzyme
- Holoenzyme
- Core enzyme consists of a^2BB’w
- Holoenzyme is the core enzyme with sigma subunit attached
Give the functions of the subunits of RNA polymerase
- Beta: Synthesises phosphodiester bonds
- Beta’: DNA binding
- Sigma: Specificity i.e. promoter recognition
- Omega: Assembly
How does the formation of a holoenzyme affect promoter binding?
The RNA polymerase enzyme core can bind anywhere on the DNA template; when the sigma subunit binds….
- The affinity of RNA polymerase for non-promoter DNA is greatly reduced
- The affinity for promoters (specific) DNA is concomitantly increased
When the holoenzyme binds DNA, how does it find the promoter sequence?
- Scans via ‘diffusion search’
- This is aided by the unusual ‘crab claw’ structure of the enzyme, which is more open when the sigma subunit binds (ie. holoenzyme formed)
Where in the promoter does bacterial RNA polymerase bind?
What is the relevance of these positions?
Which domains bind where?
- Aligns with well conserved -35 (binds domain 4 of the sigma factor) and -10 (binds domains 2 and 3) sequences; relative to start of transcription
- Since these two positions are asymmetric, this provides directionality for the enzyme to bind in the correct orientation
How are sigma factors specific?
… Give an example (E.coli)
Different classes of gene have different promoters; as such they require different sigma factors to facilitate promoter recognition
e.g. Heat shock genes require sigma ^32 instead of the ‘housekeeping’ sigma^70
e.g. Nitrogen Starvation: sigma^54
How do sigma factors recognise promoter regions?
- Via their consensus sequence
- As such, an enzyme containing a particular sigma factor can only recognise its own set of promoters
- Consensus sequences are at the same location relative to the start-point; show conserved sequences around -35 to -10, with the notable exception of sigma^54 which recognises different spacing between the two regions
+ What is the role of sigma^E?
How is it induced?
- Heat shock for extreme temperature shifts
- Induced by accumulation of unfolded (i.e. denatured) proteins in periplasm (RseA; an antisigma factor)
Sigma factor control of initiation in B.subtilis
- At least 10 sigma factors known
- Some present in vegetative cells; others only on phage infection or change from vegetative growth -> sporulation
- Major RNA pol has same struct. as E.coli
- Housekeeping: sigma^43/A
Endospore formation…
See slide 18/19
+ How do sigma factors control bacteriophage gene expression during infection of B.subtilis by SPO1 phages
Cascade of sigma-factors
- Upon infection, early genes transcribed using housekeeping s-factor gp43
- After 4-5 mins, expr. of early genes ceases, middle genes transcribed (inc. gp33, gp34)
- at 8-12 mins, transcription of middle genes replaced by late genes
Give detail of the stages of transcription
- Holoenzyme scans along -> promoter found
- RNA P docks with promoter (closed-promoter complex formed)
- Around 15-17bp of the duplex locally unwound -> open-promoter complex
- b-subunit of RNA P starts synthesising new strand of RNA -> at around 7-8 nts, sigma dissociates
- Elongation proceeds until a transcriptional termination signal is encountered
How do Rho-independent terminators work (structurally speaking)
- a.k.a intrinsic terminators
- Characterised by formation of GC-rich stem-loop (hairpin) structure followed by a run of U’s at the end of the transcribed message
- The strong pairing of the GC rich region within the RNA itself rather than the template, followed by the weak pairing of the template w/ the U-rich region probably causes the elongation complex to fall apart
