sigma factor and transcription initiation by bacterial RNAP holoenzyme Flashcards
where does the DNA enter the holoenzyme?
through the primary channel between B and B’ subunits
why is the RNAP so large when the active site is so small?
it has other functions aside from transcription -
DNA binding, DNA melting, proofreading and regulation
how is non-specific binding of the DNA prevented?
most of the RNAP is negatively charged on the surface, only the BB’ jaws are positively charged due to the presence of lysines
what is the role of the beta subunit?
holds the coding strand out of the way in the transcription bubble and holds RNA transcript by the flap domain
what is the role of the beta prime subunit?
holds the template strand in the transcription bubble, holds magnesium in the active site, and carries out NTP addition
what is the structure of sigma factor?
four alpha helical domains joined by flexible linkers
why are there no promoters in the bacterial genome that are identical to the consensus sequence?
if they were, transcription would be constitutively active
why are hexamers the most common recognition sequences for DNA?
the pitch of the helix is ten - it is difficult for a DNA binding protein to recognise more than 10 bases
why are defects deliberately inserted into promoter sequences?
to decrease promoter efficiency and allow for regulation of transcription
what are the seven steps of transcription?
1) promoter search
2) promoter binding and formation of a closed complex
3) nucleation of melting and DNA bending
4) promoter melting and formation of an open complex
5) synthesis of the first phosphodiester bond (initiation)
6) DNA scrunching
7) promoter escape and elongation
what happens during promoter search?
the RNAP interacts with DNA until the right sequence s recognised by sigma
what happens during promoter binding?
promoter at -35 is recognised by sigma via the helix-turn-helix motif
this allows it to bind in the major groove, steered in by electrostatic interactions
what happens during nucleation of melting?
when adenine at position -11 flips out due to brownian motion, the DNA becomes flexible and easy to bend
what happens during DNA bending?
sigma region 2 captures a flipped base at position 11, DNA bends around the base due to thermal fluctuations
this allows the template strand to approach the active site of the enzyme
what happens during formation of the open complex?
floppy DNA is guided into the jaws of the BB’ polymerase by positively charged amino acids
the template strand is positioned towards the active site, and the coding strand is moved out of the way
