RNA transcription Flashcards
NAC - the basics
- translocation
- NTP binding and release
- Mg2+ catalysis
- PPi binding and release
NAC - the basics
1) translocation
2) NTP binding and release
3) Mg2+ catalysis
4) PPi binding and release
translocation
transcription machinery positions dna template against mrna
catalytic phosphoryl transfer occurs by
nucleotide addition
reverse reaction
phosphorolysis
proofreading
- recovery from backtrack arrest
- exo/endonuclease hydrolysis
transcription
information to function
RNAP topology
- DNA enters pore in jaw region
- slides bridge -> wall
- 90degree reorientation
- escape channel directing
- dsDNA rudder insertion; opens
- Mg2+ catalysis
RNAP components:
stalk, clamp, jaw, duplex DNA-binding channel, catalytic centre, wall, assembly platform, rudder
upstream dna
between clamp and rudder
rna exit
rudder
RNAPs
- common accessory elongation factors (elongation-first hypothesis)
- domain-specific initiation factors
T7
- single-subunit phage RNAP
- transcribed without additional factors
- regulates bacterial expression
- co-opted for PCR
- specific promotor
Bacterial RNAP
- AT initiator affinity
beta
downstream
beta prime
central
sigma
-35, -10 regions
- transiently regulated
finding a promotor
experimental and modelling approaches
intersegmental transfer occurs when
contact is indirect
if a promotor is occupied by sigma but not RNAP
- delay/inhibition to transcription
- full contact necessary for specific binding
protection assay
- incubate RNAP w DNA
- add endo+ exo nucleases
- sequence associated with RNAP
- electrophorese for non-fragmented regions
How do sigma factors work?
- alpha-helices expose necessary aas at intervals of 3s
sigma factor
- variable, high no
FecI
don’t yet know the sequence for
How many sigma factors does e coli have
4
How many sigma factors does Bacillus obscillus have?
approx 20
How do conformational changes affect sigma factor binding?
- oxidative stress
- phosphorylation
- increase or decrease affinity
closed TI complex
dsDNA, no downstream-RNAP
intermediate TI complex
- sigma region 1.1 moves out of main channel
- downstream dna moves in
open TI complex
- dna strands separate
- template strand bps with first NTP
scrunching
production of >10nt oligos
Ds/ss DNA-sigma interactions
A-11 and T-7 flip, resulting in dsDNA separation via melting
supercoils
torsional tension
high DNA-DNA interaction
transcriptional quiescence
low DNA-DNA interaction
heavy transcription
RNAPI
18s/28s rRNA
RNAPII
- mRNA
- some sRNAs
RNAPIII
- tRNAs
- 5S rRNAs
- other sRNAs
RNAPIV
inverted dna repeat transcription in RNAi-mediated DNA methylation; transcribes ONSEN
eukaryotic initiation
requires basal TFs to generate docking sites
RNAPI initiation
- UBF binds upstream promotor
- SL1 binds AT-rich core promotor
SL1
basal TF
RNAPII initiation
- TATA BP binds
- DNA bends up to 80degrees
- TFTA, TFTB associate
- RNAP recruitment
- TF2E, TF2F, TF2H
TF2F
catalytic and general TF
TF23, H
- separate strands
- energy consumptive
RNAPIII initiation
- 3 different promotors; conserved elements in alternative configurations
Elongation
- TF2H kinase phosphorylate catalytic subunit C-terminal domains
- factor release and recycling
Termination
- CstF and CpsF bind to catalytic subunit C-terminal domain
- CstF cuts terminator
- CpsF recruits alternative polymerases for poly-A
- RNAP dissociates
CstF
- capping specification termination factor
- endonuclease
CpsF
capping and polyadenylation specification factor
polyA
100-250bps
Two end-termination hypotheses
- catalytic subunit confirmation change causes loss of affinity
- catalytic subunit displaced by uncapped exonuclease action
moderate transcription
transient H2A/B loss/exchange
active transcription
transient octamer loss/exchange
NAC - the specifics
- nucleophilic free hydroxide attacks and captures 1st nucleotide C3 OH proton
- cascade
- C2O links covalently to C5PO43-
- associates w/ proton acceptor
- free to diffuse
What is NAC?
phosphor transfer reaction
