RNA Polymerase And Transcription Flashcards
at what level is gene expression controlled
transcription level, turning genes on and off
what are the phases of transcription
initiation, elongation and termination
what is a holoenzyme
an RNA polymerase enzyme which is associated with a sigma factor
explain closed complex
the reversible first stage on initiation. the sigma is bound to RNAP which allows location of the promoter and the DNA duplex is still closed
explain open complex
the irreversible second stage of initiation where mRNA transcription begins and DNA unwinds
explain abortive initiation
the third stage of initiation. around 10bp are transcribed but it can’t go further as the sigma factor is still bound to the promoter
explain processive initiation
the final stage of initiation. sigma is ejected which allows polymerase to form a ‘crab claw’ on DNA and elongate mRNA, locating it in the exit channel
what is RNAP composed of
two alpha subunits, a beta, a beta prime, an omega, and this is associated with a sigma factor
how does sigma bind to the primer
it has two projecting lobes- sigma 2 binds to -10 and sigma 4 binds to -35
where does the DNA break first in the open complex
at the -10 region, it is rich in AT and therefore the bonds are weaker
why is sigma ejected
two reasons- it prevents the elongation of mRNA beyond 10bps and and it blocks the exit channel
where does recognition of the promoter occur
the -35 region of the promoter and the sigma 4 lobe via direct or indirect readout
what does 6S RNS do
it accumulates in the stationary phase and mimics the open complex, preventing transcription. it dissociates with the increase of NTPs
explain catalysis
the previous nucleotide is translocated to the i region to free up the i+1 region. the new NTP is hydrogen bonded to the exposed base of the DNA template, then a phosphodiester bond to the previous nucleotide forms, releasing the resultant PPi
how is transcriptional accuracy ensured
two Mg2+ ions and the trigger loop/ helix are involves. one Mg2+ is associated with the core and one with the incoming NTP, if it is the right one the trigger helix pushed it into place next to the first ion, allowing catalysis to happen
what interrupts transcription
antibiotics or DNA templates with gaps in can cause stalling or arrest
what are the two types of termination
intrinsic (the sequence forms hairpin loops which open the exit) and Rho dependent (Rho proteins disengage the RNAP from the mRNA
what are the two important sigma variations
sigma 70, expressed in exponential phase, and sigma 38, expressed in stationary phase
how is the expression of sigma 38 changed
molecules can upregulate it (eg ppGpp) or downregulate it (eg cyclic AMP), they both alter expression of rpoS gene which encodes sigma 38
what is ClpP
a protease responsible for sigma 38 degradation- activity reduces in stationary phase to allow competition with sigma 70
what is the stringent response
reaction to amino acid starvation- works by inhibiting transcription of translation machinery eg tRNA, and by enhancing transcription of amino acid biosynthesis genes
how does control of genes work
regulation is intrinsic to the DNA of the promoter region of the controlled gene- both negative or positive regulation
describe negative control
the CC region of the G rich discriminator in the non- template strand binds to ppGpp, taking with it a Mg2+ ion which prevents catalysis and also tightens the RNAP-DNA complex
describe positive control
the discriminator in the non- template strand is AT rich so ppGpp can’t bind, catalysis can’t be stopped and the RNAP-DNA complex is loosened. it also upregulates transcription to produce sigma 38, stimulating transcription of more stress genes
what is DksA
a protein which binds to the RNAP on the secondary channel and traps the trigger loop, preventing catalysis
how is DNA repaired
ppGpp repairs DNA by base excision repair
what is phase variation
changing whether the expression of a gene is on or off depending on which direction a promoter is flipped
what type of promoters does phase variation affect
divergent promoters which alternate in direction
explain phase variation in Salmonella
fliC encodes H2 flagellin and fliB encodes H1- one on either side of the promoter and inverting region. rh1 encodes a repressor which prevents the transcription of H1 when promotion is in the direction of H2
what is the structure of the inverting region
it is a Hin gene with two hix sites, one on either side, and inversion is stimulated by the binding of proteins Fis and HU
why is phase variation important
it is significant in the immune response and environmental response (eg temperature change)
