prokaryotic transcription Flashcards
promoter =
sequence of DNA that initiates transcription of a particular gene
first base to be copied into mRNA is the last base in the promoter sequence
terminator =
sequence of DNA that marks the end of agene/operon
last base to be copied into mRNA is the last base in the terminator sequence
RNA polymerase =
enzyme which uses a single-stranded DNA template to synthesise a complementary strand (in the 5’ to 3’ direction)
initiation of transcription =
promoter binding and promoter melting by RNA pol
- RNA pol binds to the promoter with single base pair precision
- RNA pol separates the DNA strands, providing a single-stranded template
elongation of transcription =
making of mRNA by RNA pol
- template strand of DNA is read by RNA pol and complementary nucleotides are used to build mRNA (with the same sequence as the coding strand but with Uracil instead of Thymine)
- chain grows from 5’ to 3’
termination of transcription =
dissociation of RNA pol + mRNA from DNA
- terminator signals that the RNA transcript is complete
- transcript is released from the RNA pol
how RNA polymerase binds to the promoter…
- RNA pol reads the sequence before melting (normally finds promoter in the major groove)
- at physiological temperatures, DNA is constantly affected by BROWNIAN MOTION = erratic random movement of particle as a result of continuous bombardment from surrounding molecules = causes occasional base flipping in/out but not breaking of strands
- hydrogen bonds holding bases together can open spontaneously to allow RNA pol to bind but covalent phosphodiester bonds do not break
can detect transcription…
using nucleoside triphosphates containing radioactive alpha phosphate = RNA backbone (phosphodiester bonds) becomes radioactive
why is initiation the most regulated process…
transcription doesn’t require a primer - instead relies on accurate binding of RNA pol (single base pair precision)
transcription factors regulate RNA pol binding
prokaryotic and eukaryotic transcription work by the same basic principles, but IN PROKARYOTES…
- transcription and translation are coupled (no nucleus)
- DNA is not packaged into histones = DNA is accessible to RNA pol
- genes are often combined into groups (operons) = genes are regulated by one promoter = one mRNA is polycistronic (encodes for several polypeptides)
- Shine-Dalgarno
- mRNA is not processed = no splicing (as prokaryotes don’t have introns), no capping of the 5’ end and no polyadenylation of the 3’ end
Shine-Dalgarno =
a ribosomal binding site in prokaryotic mRNA located 8 bases upstream of thee start codon AUG
- helps recruit the ribosome to the mRNA to initiate protein synthesis by aligning it with the start codon
how is RNA pol discovered…
in vitro transcription assay
1) make cell lysate - centrifuge prokaryote to collect cells, then add lysozyme for cell lysis, centrifuge to get rid of cell debris and DNA - leaving the cell extract containing RNA pol
2) invent a transcription assay - add nucleoside triphosphates (ATP, UTP, CTP, GTP), radioactive alpha phosphate and DNA template to the lysate = makes radioactive labelled RNA
3) purify - use the transcription assays and chromatography to isolate bacterial RNA pol from lysate (collect protein fractions)
4) find which fraction contains RNAP by seeing which fraction has radioactive labelled RNA
5) check by running fraction on SDS-PAGE = RNA pol has 4 bands (4 types of polypeptides)
prokaryotic RNA pol core:
made up of 5 subunits ( two α subunits, a β subunit, a β′ subunit and a ω subunit)
DNA helix enters through a primary channel between β and β′
nucleoside triphosphates enter through a secondary channel formed by β′
in the active site, Mg2+ is held in place with ionic bonds with 3 aspartate residues
what happens in the RNAP core during initiation…
DNA makes a 90-degree turn inside the core by bouncing against a ‘wall’ created by the β′ subunit = this creates a transcription bubble when the DNA melts (unwinds)
functions of the beta prime (β′) subunit -
does most of the work in elongation…
- nucleoside triphosphate addition through a secondary channel
- holds the Mg2+ in the active site with ionic bonds with 3 aspartate residues
- holds the template strand in the transcription bubble
- creates a wall to bend the DNA 90-degrees
functions of the beta (β) subunit -
- holds the coding strand in the transcription bubble up and out of the way
- flap domain forms an exit channel for RNA = forms a ring around the RNA
- antibiotic resistance = some mutations in the beta subunit make RNA pol resistant to binding of antibodies
importance of RNA exit channel and crab claw shape of RNAP core -
keep RNA clamped on DNA
functions of the alpha 1 and 2 (α) subunits -
assembly of RNAP
promoter recognition
function of the omega (ω) subunit -
helps β′ fold correctly
sigma factor:
core RNAP need sigma factor ( a transcription factor) to bind and melt promoters to initiate transcription
required for accurate transciption initiation
core RNAP + sigma factor = RNAP holoenzyme
sigma is comprised of:
four alpha-helical domains joined together by flexible linkers
these domains are re-lined up between the jaws of RNAP on the back side of RNAP (facing upstream)
key steps of prokaryotic transcription initiation by RNAP holoenzyme:
1) promoter search
2) promoter binding (formation of closed complex)
3) nucleation of melting
4) DNA bending
5) promoter melting (formation of open complex)
6) synthesis of the first phosphodiester bond (initiation)
7) DNA scrunching
8) promoter escape and elongation
step 1 of prokaryotic transcription initiation by RNAP holoenzyme: PROMOTER SEARCHING
RNAP aimlessly diffuses in 3D until it accidentally bumps into a random DNA segment = slow
RNAP then reads the DNA major groove in 1D with its sigma domain = speeded up by facilitated diffusion
reads without melting the DNA until it finds the promoter
step 2 of prokaryotic transcription initiation by RNAP holoenzyme: PROMOTER BINDING
closed complex formation
promoter element is recognised by the helix-turn-helix of sigma domain 4 - promoter binds in the major groove of the hexameric promoter DNA sequence
DNA is not yet melted
step 3 of prokaryotic transcription initiation by RNAP holoenzyme: NUCLEATION OF PROMOTER MELTING
adenine is the nucleation base
thermal fluctuations cause the adneine base to flip out of the DNA helix and get captured in the hydrophobic pocket
step 4 of prokaryotic transcription initiation by RNAP holoenzyme: DNA BENDING
sigma region 2 captures a flipped base and DNA bends around this flipped base by thermal fluctuations - the DNA ends up between the two jaws of RNA pol
step 5 of prokaryotic transcription initiation by RNAP holoenzyme: FORMATION OF ‘OPEN COMPLEX’
promoter unwinding/melting = template and coding strands separate = transcription bubble is formed
step 6 of prokaryotic transcription initiation by RNAP holoenzyme: INITIATION
synthesis of the first phophodiester bond
step 7 of prokaryotic transcription initiation by RNAP holoenzyme: DNA ‘SCRUNCHING’
ssDNA strand gets pulled inside the RNA pol cleft, like two coiled springs
5’ end of nascent RNA cannot get out because sigma domain 4 is stuck in the RNA exit channel
takes a long time for RNA pol to leave the promoter (takes many attempts)
step 8 of prokaryotic transcription initiation by RNAP holoenzyme: PROMOTER ESCAPE AND ELONGATION
after many attempts, RNA pol kicks out sigma domain 4 with the growing RNA 5’ end and escapes the promoter
why is it difficult for RNAP to escape the promoter:
RNAPs do not use primers. So, the RNAP holenzyme has to bind to promoters very tightly, to align its active site precisely
Tight interactions take energy and time to break
It often takes RNAP 100+ failed attempts to escape the promoter
(“abortive initiation”) and commit to elongation of RNA
The energy for escape is accumulated in the scrunched state (in which the DNA is pulled inside and wound up like a spring), and is then released in a single powerful burst, launching RNAP down the DNA
prokaryotic transcription initiation by RNAP holoenzyme can be blocked by…
…antibiotic rifampicin (anti-tuberculosis antibiotic) - binds to the beta subunit of RNA pol (inside RNA exit channel) an prevents formation of transcription longer than 2 nucleotides - bacteria can acquire resistance with due to mutation
