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| Poly-A retrotransposons |
uses the Pol III-dependent transcription |
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mcs5109 Wed, 19 Nov 2008 22:52:25 GMT |
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| Pol III promoter trans-elements |
TFIIIC recruits TFIIIB (TBP+Brf+Bdp) to a promoter then TFIIIB recruits Pol III to a promoter |
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mcs5109 Wed, 19 Nov 2008 22:52:25 GMT |
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| Pol III promoter cis-elements |
BoxA & BoxB (TFIIIC binding site) |
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mcs5109 Wed, 19 Nov 2008 22:52:25 GMT |
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| Pol I promoter trans-elements |
-UBF & SL1 (contains TBP)
-recruits Pol I to increase activity. |
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mcs5109 Wed, 19 Nov 2008 22:52:25 GMT |
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| Pol I promoter cis-elements |
1. Core promoter (Pol I binding site)
2. UCE (Upstream Control Element, trans-element binding site) |
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mcs5109 Wed, 19 Nov 2008 22:52:25 GMT |
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| RNase |
-in torpedo model
-chews uncapped end until PolyA tail
-Pol II dissociates |
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mcs5109 Wed, 19 Nov 2008 22:52:25 GMT |
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| Poly-A polymerase |
adds ~200 As to the RNA’s 3’ end (w/o template) |
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mcs5109 Wed, 19 Nov 2008 22:52:25 GMT |
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| Polyadenylation |
-termination
-Pol II CTD recruits PolyA factors
-Poly-A polymerase: adds ~200 As to the RNA’s 3’ end (w/o template) |
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mcs5109 Wed, 19 Nov 2008 22:52:25 GMT |
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| FACT |
-FAcilitates Chromatin Transcription
-heterodimer of Spt16 and SSRP1
-removes H2A/H2B dimer |
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mcs5109 Wed, 19 Nov 2008 22:28:06 GMT |
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| TFIIS |
limits the length of time RNAP pauses; stimulates hydrolysis proofreading
-analygous to GreB in pros |
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mcs5109 Wed, 19 Nov 2008 22:28:06 GMT |
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| ELL |
-increases elongation rate |
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mcs5109 Wed, 19 Nov 2008 22:28:06 GMT |
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| P-TEFb |
Positive Transcription Elongation Factor b
-cyclin-dependent
-phosphoryates CTD of Pol II
-ELL, TFIIS |
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mcs5109 Wed, 19 Nov 2008 22:28:06 GMT |
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| activators |
-interact w/ diff mediator subunits |
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mcs5109 Wed, 19 Nov 2008 22:15:38 GMT |
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| 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 |
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mcs5109 Wed, 19 Nov 2008 22:15:38 GMT |
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| TFIIH |
Melts promoter
MW similar to Pol II
nt mismatch repair |
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mcs5109 Wed, 19 Nov 2008 22:05:01 GMT |
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| TFIIE |
Recruits TFIIH |
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mcs5109 Wed, 19 Nov 2008 22:05:01 GMT |
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| TFIIF |
Binds to Pol II
Similar to s subunit of bacterial RNAP
stabilize open complex |
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mcs5109 Wed, 19 Nov 2008 22:05:01 GMT |
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| 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 |
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mcs5109 Wed, 19 Nov 2008 22:05:01 GMT |
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| SAGA |
-histone modification enzyme
-associated w/ some TAFs |
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mcs5109 Wed, 19 Nov 2008 22:05:01 GMT |
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| 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). |
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mcs5109 Wed, 19 Nov 2008 22:05:01 GMT |
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| How TBP distorts DNA |
-TBP binds in the minor groove of DNA at the TATA box
-bends DNA about 80 degrees |
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mcs5109 Wed, 19 Nov 2008 22:05:01 GMT |
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| GTFs of Pol II |
-increasing # of subunits |
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mcs5109 Wed, 19 Nov 2008 21:29:44 GMT |
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| 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) |
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mcs5109 Wed, 19 Nov 2008 21:27:09 GMT |
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| Pol II Initiation |
-CTD must be unphosphoylated
CDT repeats: Tyr-Ser-Pro-Thr-Ser-Pro-Ser |
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mcs5109 Wed, 19 Nov 2008 21:27:09 GMT |
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| TFIIH |
-promoter melting |
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mcs5109 Wed, 19 Nov 2008 21:27:09 GMT |
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| 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 |
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mcs5109 Wed, 19 Nov 2008 21:25:13 GMT |
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| Pre-initiation complex |
all general transcription factors and polymerase bound at promoter |
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mcs5109 Wed, 19 Nov 2008 21:25:13 GMT |
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| in vivo promoters |
Upstream (usually) of the core promoter contains other elements required for efficient transcription
-can be 100kbs away up or downstream |
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mcs5109 Wed, 19 Nov 2008 21:25:13 GMT |
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| DCE and DPE |
downstream promoter elements |
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mcs5109 Wed, 19 Nov 2008 21:17:35 GMT |
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| Inr |
initiator |
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mcs5109 Wed, 19 Nov 2008 21:17:35 GMT |
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| TATA box |
TBP recognition element |
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mcs5109 Wed, 19 Nov 2008 21:17:35 GMT |
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| BRE |
Define trx orientation
TFIIB recognition element |
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mcs5109 Wed, 19 Nov 2008 21:17:35 GMT |
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| RNAP II Core Promoters |
1. BRE
2. TATA box
3. Inr
4. DPE or DCE
usually only has 2-3 per promoter |
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mcs5109 Wed, 19 Nov 2008 21:18:41 GMT |
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| rut site |
-40 nts w/o secondary structure
-rho utilization site
-rich in C
-recruits Rho to the transcribing RNA |
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mcs5109 Wed, 19 Nov 2008 21:14:58 GMT |
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| 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 |
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mcs5109 Wed, 19 Nov 2008 21:14:58 GMT |
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| 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 |
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mcs5109 Wed, 19 Nov 2008 21:14:58 GMT |
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| Single-Subunit RNA Polymerases |
-Bacteriophage, chloroplast and mitochondria
-T7-phage |
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mcs5109 Wed, 19 Nov 2008 21:14:58 GMT |
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| TFIIS |
-elongation factor in euks
-provides Mg++
-hydrolysis editing |
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mcs5109 Wed, 19 Nov 2008 20:56:11 GMT |
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| Gre |
-elongation factor in bacteria
-provides Mg++
-hydrolysis editing |
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mcs5109 Wed, 19 Nov 2008 20:56:11 GMT |
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| Is there 3'--> 5' nuclease activity in RNAP? |
No |
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mcs5109 Wed, 19 Nov 2008 20:56:11 GMT |
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| 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 |
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mcs5109 Wed, 19 Nov 2008 20:56:11 GMT |
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| pyrophosphorolytic editing |
a simple back-reaction of RNA synthesis, requires pyrophosphate (PPi) |
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mcs5109 Wed, 19 Nov 2008 20:56:11 GMT |
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| Elongating process |
~8 bp DNA/RNA hybrid in the active site
Adds to 3’ site
RNAP synthesizes and proofreads at active site |
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mcs5109 Wed, 19 Nov 2008 20:52:42 GMT |
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| What leaves before elongation? |
bacteria: sigma subunit
euk: only TFIID and TFIIA stay at promoter |
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mcs5109 Wed, 19 Nov 2008 20:52:42 GMT |
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| 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) |
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mcs5109 Wed, 19 Nov 2008 20:52:42 GMT |
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| How is non-template ssDNA stabilized? |
sigma 2 |
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mcs5109 Wed, 19 Nov 2008 20:52:42 GMT |
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| Is OCF temp-dependent? |
Yes. |
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mcs5109 Wed, 19 Nov 2008 20:43:01 GMT |
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| Kf |
-kinetics constant |
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mcs5109 Wed, 19 Nov 2008 20:43:01 GMT |
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| Kb |
-binding constant
-promoter strength |
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mcs5109 Wed, 19 Nov 2008 20:43:01 GMT |
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| Model of initial RNAP transcription |
-transient
-inchworming
-scrunching |
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mcs5109 Wed, 19 Nov 2008 20:43:01 GMT |
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| sigma 4 |
-35 element recognition by helix-turn-helix motif |
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mcs5109 Wed, 19 Nov 2008 20:37:36 GMT |
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| sigma 3 |
extended -10 element recognition |
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mcs5109 Wed, 19 Nov 2008 20:37:36 GMT |
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| sigma 2 |
-10 element recognition and DNA melting |
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mcs5109 Wed, 19 Nov 2008 20:37:36 GMT |
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| UP element |
before -35 sequence
-recognized by carboxyl terminal domains of the a subunit
-"elephant trunk" |
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mcs5109 Wed, 19 Nov 2008 20:36:46 GMT |
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| extended -10 |
-only if no -35 |
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mcs5109 Wed, 19 Nov 2008 20:35:17 GMT |
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| 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 |
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mcs5109 Wed, 19 Nov 2008 20:35:17 GMT |
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| TBP and TFB |
-GTFs required for archae RNAP
-TFB is a TFIIB homologue |
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mcs5109 Wed, 19 Nov 2008 20:28:49 GMT |
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| Archae RNAP |
-synthesizes m, r, t
-most similar to Pol II sequence-wise
-require GTFs: TBP and TFB
-no known inhibitors |
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mcs5109 Wed, 19 Nov 2008 20:28:49 GMT |
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| 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 |
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mcs5109 Wed, 19 Nov 2008 20:28:49 GMT |
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| MW of euk RNAPs |
500 kDa |
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mcs5109 Wed, 19 Nov 2008 20:17:56 GMT |
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| Pol III |
-tRNA
-less sensitive to a-amanitin |
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mcs5109 Wed, 19 Nov 2008 20:17:56 GMT |
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| Pol II |
-mRNA
-very sensitive to a-amanitin |
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mcs5109 Wed, 19 Nov 2008 20:17:56 GMT |
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| Pol I |
-rRNA
-insensitive to a-amanitin |
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mcs5109 Wed, 19 Nov 2008 20:17:56 GMT |
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| Rifampicin |
-anti-tuberculosis treatment, blocks RNA extension |
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mcs5109 Wed, 19 Nov 2008 20:15:40 GMT |
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| BB' subunit of bacterial RNAP |
-pincers
-active site at center |
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mcs5109 Wed, 19 Nov 2008 20:15:40 GMT |
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| Holoenzyme |
-core + s subunit
-start RNA synthesis at promoter
-s determines promoter specificity |
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mcs5109 Wed, 19 Nov 2008 20:15:40 GMT |
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| 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 |
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mcs5109 Wed, 19 Nov 2008 20:15:40 GMT |
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| bacterial RNAP subunits |
-conserved among all cellular organisms |
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mcs5109 Wed, 19 Nov 2008 20:08:57 GMT |
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| RPB6 |
-subunit conserved in Pol I, II, III |
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mcs5109 Wed, 19 Nov 2008 20:08:57 GMT |
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| de novo transcription |
-RNA synthesis usually doesn't require a primer |
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mcs5109 Wed, 19 Nov 2008 20:08:56 GMT |
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| Does RNA synthesis require a primer? |
NO!
de novo |
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mcs5109 Wed, 19 Nov 2008 20:08:56 GMT |
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