Study New BMB 400 Chap 12 Flash Cards

 
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New BMB 400 Chap 12

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Poly-A retrotransposons
uses the Pol III-dependent transcription
Pol III promoter trans-elements
TFIIIC recruits TFIIIB (TBP+Brf+Bdp) to a promoter then TFIIIB recruits Pol III to a promoter
Pol III promoter cis-elements
BoxA & BoxB (TFIIIC binding site)
Pol I promoter trans-elements
-UBF & SL1 (contains TBP)
-recruits Pol I to increase activity.
Pol I promoter cis-elements
1. Core promoter (Pol I binding site)
2. UCE (Upstream Control Element, trans-element binding site)
RNase
-in torpedo model
-chews uncapped end until PolyA tail
-Pol II dissociates
Poly-A polymerase
adds ~200 As to the RNA’s 3’ end (w/o template)
Polyadenylation
-termination
-Pol II CTD recruits PolyA factors
-Poly-A polymerase: adds ~200 As to the RNA’s 3’ end (w/o template)
FACT
-FAcilitates Chromatin Transcription
-heterodimer of Spt16 and SSRP1
-removes H2A/H2B dimer
TFIIS
limits the length of time RNAP pauses; stimulates hydrolysis proofreading
-analygous to GreB in pros
ELL
-increases elongation rate
P-TEFb
Positive Transcription Elongation Factor b
-cyclin-dependent
-phosphoryates CTD of Pol II
-ELL, TFIIS
activators
-interact w/ diff mediator subunits
mediator complex
-b/w CDT of Pol II and activators
-Deletion of diff subunits of Mediator leads to loss of diff genes
-only mediator responds to activator
-20 subunits
-various forms w/ diff subunits
TFIIH
Melts promoter
MW similar to Pol II
nt mismatch repair
TFIIE
Recruits TFIIH
TFIIF
Binds to Pol II
Similar to s subunit of bacterial RNAP
stabilize open complex
TFIIB
Bridge b/w TBP and RNAP Pol II
Determines polarity
NTD inserts into the RNA exit channel of Pol II
supports NTP binding for transcription initiation
SAGA
-histone modification enzyme
-associated w/ some TAFs
TAFs
TBP-associated factors
-bind DNA elements at the promoter (Inr and DPE)
-Similar to histone structure
-associated with some histone modification enzymes (e.g., SAGA).
How TBP distorts DNA
-TBP binds in the minor groove of DNA at the TATA box
-bends DNA about 80 degrees
GTFs of Pol II
-increasing # of subunits
Pol II Elongation
-CTD must be phosphoylated
-Phosphorylated CTD provides the binding sites for auxiliary factors (e.g., for 5’ cap, 3’ Poly A tail)
Pol II Initiation
-CTD must be unphosphoylated

CDT repeats: Tyr-Ser-Pro-Thr-Ser-Pro-Ser
TFIIH
-promoter melting
General transcription factors
required for specific transcription
Not subunits of purified RNAPs
Required to bind to promoters
GTFs for Pol II are called TFIIx, where x = A, B, D, …
Can have multiple subunits
Pre-initiation complex
all general transcription factors and polymerase bound at promoter
in vivo promoters
Upstream (usually) of the core promoter contains other elements required for efficient transcription
-can be 100kbs away up or downstream
RNAP II Core Promoters
1. BRE
2. TATA box
3. Inr
4. DPE or DCE

usually only has 2-3 per promoter
DCE and DPE
downstream promoter elements
Inr
initiator
TATA box
TBP recognition element
BRE
Define trx orientation
TFIIB recognition element
rut site
-40 nts w/o secondary structure
-rho utilization site
-rich in C
-recruits Rho to the transcribing RNA
rho-dependent
-Rho is a hexamer ATPase
-Rho binds to RNA and moves along it
-If reaches a paused RNAP, removes RNAP and unwinds the RNA-DNA duplex, using ATP hydrolysis
Only terminates at end of gene/operon
Rho-independent
-GC region with a palindromic sequence
-4 to 10 consecutive A’s on template strand
-GC= hairpin
-U’s downstream
-Hairpin either:
forces open RNA exit channel (steric clash model)
disengage RNA 3’-OH from the active center (allosteric model).
-A:U base pairs are the weakest of all base pairs
-easily disrupted by hairpin
Single-Subunit RNA Polymerases
-Bacteriophage, chloroplast and mitochondria
-T7-phage
TFIIS
-elongation factor in euks
-provides Mg++
-hydrolysis editing
Gre
-elongation factor in bacteria
-provides Mg++
-hydrolysis editing
Is there 3'--> 5' nuclease activity in RNAP?
No
hydrolysis editing
-RNAP backtracks by one or more nt
-removes error-containing sequence
-stimulated by Gre (in bacteria) and TFIIS (in eukaryote)
-factors provide Mg++ at the active center for rxn
pyrophosphorolytic editing
a simple back-reaction of RNA synthesis, requires pyrophosphate (PPi)
Elongating process
~8 bp DNA/RNA hybrid in the active site
Adds to 3’ site
RNAP synthesizes and proofreads at active site
What leaves before elongation?
bacteria: sigma subunit
euk: only TFIID and TFIIA stay at promoter
starting out transcription
-regulated by [nt]
-need extra nts to make 1st phosphodiester bond
-Extra interactions: bacterial Sigma subunit (region 3.2) or euk TFIIB (B-finger)
How is non-template ssDNA stabilized?
sigma 2
Is OCF temp-dependent?
Yes.
Kf
-kinetics constant
Kb
-binding constant
-promoter strength
Model of initial RNAP transcription
-transient
-inchworming
-scrunching
sigma 4
-35 element recognition by helix-turn-helix motif
sigma 3
extended -10 element recognition
sigma 2
-10 element recognition and DNA melting
UP element
before -35 sequence
-recognized by carboxyl terminal domains of the a subunit
-"elephant trunk"
extended -10
-only if no -35
Bacterial promoter features
-10: TATAAT
-35: TTGACA
-17~19 bp between -10 and -35 elements
-recognized by s subunit
-RNAP binding site: -60 to +20
-also, extended -10 and UP
TBP and TFB
-GTFs required for archae RNAP
-TFB is a TFIIB homologue
Archae RNAP
-synthesizes m, r, t
-most similar to Pol II sequence-wise
-require GTFs: TBP and TFB
-no known inhibitors
a-Amanitin
-Mitochondrial, chloroplast, archaea and prokaryotic RNAPs are insensitive
-from death cap
-Takes a while to die if you eat it: from slow turnover of mRNAs
MW of euk RNAPs
500 kDa
Pol III
-tRNA
-less sensitive to a-amanitin
Pol II
-mRNA
-very sensitive to a-amanitin
Pol I
-rRNA
-insensitive to a-amanitin
Rifampicin
-anti-tuberculosis treatment, blocks RNA extension
BB' subunit of bacterial RNAP
-pincers
-active site at center
Holoenzyme
-core + s subunit
-start RNA synthesis at promoter
-s determines promoter specificity
bacterial core enzyme
2 alphas, BB', W
-catalyzes RNA synthesis (m, r, t)
-BB' are 2 "pincers"
-27 A channel for dsDNA
-secondary channel for NTPs
bacterial RNAP subunits
-conserved among all cellular organisms
RPB6
-subunit conserved in Pol I, II, III
de novo transcription
-RNA synthesis usually doesn't require a primer
Does RNA synthesis require a primer?
NO!
de novo
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