How does transcription work?
• Initiation
• Elongation
• Termination
Transcription in prokaryota
α2ββ’ω = core enzyme
σ factor = holoenzyme (holoenzyme
binds stably only at promoter and start
transcription at the proper site ! sigma only
needed for initiation but when few NTs added it
detaches from the core enzyme )
different σ-factor ! different consensus sequence
in the promoter region
ω subunit facilitates assembly of RNAP and
stabilizes assembled RNAP.
Two alpha (α) subunits of 36 kDa, a beta (β) α ! assembly of polymerase and positive control of
subunit of 150 kDa, a beta prime subunit (β′) of transcription initiation + elongation
155 kDa, and a small omega (ω) subunit. β subunit ! synthesis + non-sequence-specific
interactions with DNA
• Only one type RNA polymerase for: mRNA, tRNA, rRNA
• Large multimeric unit
• 4-subunits (α2ββ’) + 1 ω (omega) ! 380 kDa
• σ factor (20-70 kDa) -7 different sigma in E. coli (housekeeping σ70, heat shock σ32)
2006 Nobel Prize in Chemistry was awarded to Roger D. Kornberg
Initiation in
prokaryota
Initiation:
• Promoter
recognition
• Formation of
transcription
bubble
• Creation of the
first bond between
rNTPs
• Escape of
transcription
apparatus from
the promoter
Transcription initiation
• Core (α2ββ’ω) binds Sigma (σ70 for house keeping gene) ! recognize
• Recognize up of promoter, binds and slides! and bind -35
and -10 relative to +1 but can bind -45 or-40 to +20 region !
form closed promoter complex.
• -35 and -10 determine the strand and direction of
transcription.
• Sometime -40 to -60 region is AT rich interact with α and
enhance transcription.
• Enzyme binds weakly but when bound change its
conformation (isomerization) and bound tightly ! unwind
dsDNA ! form open promoter complex
• Unwind start at -10 consensus and extend downstream.
• Form a transcription bubble or 12 to15 NTs (-11 to +4)
• Open promoter complex+ NTPs from Initial Transcription
complex ! 1st phosphodiester bond (+1!+2) [synthesis begin]
• Initial Transcription complex squeeze ! +2 to +15 is pulled
! bubble expansion (~25 NTs) ! 80 mer ! σ release
• Unwind start at -10
consensus and extend
downstream.
• Form a transcription
bubble or 12 to15 NTs (-11
to +4)
• 1st phosphodiester bond
(+1!+2) [synthesis begin]
• Initial Transcription
complex squeeze ! +2 to
+15 is pulled ! bubble
expansion (~25 NTs) ! 80
mer ! σ release
11-15 NTs RNA (8-9 mer in bubble as DNA-RNA hybrid! mRNA
detach ! DNA rewind) removes exit channel blockage and escape
from promoter ! elongation 40NTs/sec (RNA pol squeeze !
continue)
In abortive initiation, RNA polymerase re-winds (SCRUNCH) and
ejects the downstream portion of the unwound DNA, releasing the
RNA, and reverting to the RNA polymerase-promoter open complex.
A bacterial elongation complex showing the movement of the enzyme
along the template DNA strand from the 3ˊ towards the 5ˊ end and RNA
strand is elongated in the 5ˊ →3ˊ direction. NTPs- Nucleoside
triphosphates
28- 7
Termination
in Bacteria
ρ-dependent ρ-independent
termination termination
(Rho) ρ-independent termination:
• In RNA transcript, a sequence (7-20
base) rich in G-C base pairs form
stem-loop followed by a chain of U !
loop slow down Pol.
• NusA protein bound to RNA pol
tightly bind stem loop and temporarily
stall RNA pol.
• Chain of U-A bp weak bond ! weak
bond lower energy of destabilization
for the DNA-RNA duplex ! allow
unwinding and dissociate RNA Pol.
