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| Spt16 and SSRP1 |
-heterodimer in FACT |
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mcs5109 Wed, 19 Nov 2008 03:32:55 GMT |
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| What to do about histones in elongating Pol II? |
FACT: FAcilitates chromatin transcription
-dismantles nucelosomes
-removes H2A/H2B dimer
-comprised of heterodimer of Spt16 and SSRP1 |
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mcs5109 Wed, 19 Nov 2008 03:32:28 GMT |
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| GreB |
-bacterial enzyme acts like auk TFIIS (in Pol II) |
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mcs5109 Wed, 19 Nov 2008 03:32:28 GMT |
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| TFIIS |
-limits length of time RNAP II pauses
-proofreads, too
-misincorporated bases slows down RNAP II
-TFIIS stimulates hydrolysis activity
-releases misincorporated bases |
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mcs5109 Wed, 19 Nov 2008 03:28:28 GMT |
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| ELL |
-bind to Pol II
-suppress transient pausing
-increases elongation rate |
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mcs5109 Wed, 19 Nov 2008 03:28:28 GMT |
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| P-TEFb |
-cyclin-dependent kinase
phosphorylates CTD domain of Pol II |
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mcs5109 Wed, 19 Nov 2008 03:28:28 GMT |
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| Proteins of elongation |
-P-TEFb (protein transcription elongation factor b)
-ELL
-TFIIS |
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mcs5109 Wed, 19 Nov 2008 03:28:28 GMT |
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| what factors stay behind after initiation? |
TFIID and mediator |
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mcs5109 Wed, 19 Nov 2008 03:28:28 GMT |
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| Pol II Elongation/RNA proofreading |
-only TFIID and mediator stay behind after initiation |
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mcs5109 Wed, 19 Nov 2008 03:28:28 GMT |
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| RNA Pol II holoenzyme |
-can be isolated as single complex
-arrive at promoter in single complex? |
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mcs5109 Wed, 19 Nov 2008 03:14:46 GMT |
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| Mediator complex |
>20 subunits
-modules can dissociate from each other in certain conditions in vitro
-different forms w/ diff subunits
-regulate diff subsets of genes |
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mcs5109 Wed, 19 Nov 2008 03:14:45 GMT |
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| changes to initiation in vivo |
-in vivo, DNA packaged into chromatin
-need:
1. transcriptional regulatory proteins
2. mediator complex
3. nucleosome-modifying enzymes
-see diagram |
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mcs5109 Wed, 19 Nov 2008 03:14:45 GMT |
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| TFIIH |
-converts PIC to open complex
-9 subunits
-MW comparable to Pol II
-involved in nt mismatch repair |
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mcs5109 Wed, 19 Nov 2008 02:45:56 GMT |
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| TFIIE |
-recruits TFIIH |
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mcs5109 Wed, 19 Nov 2008 02:45:56 GMT |
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| TFIIF |
-2 subunits
-associated w/ Pol II (recruited together)
-stabilizes DNA/TBP/TFIIB complex |
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mcs5109 Wed, 19 Nov 2008 02:45:56 GMT |
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| TFIIB |
-TBF in archae
-single polypeptide chain
-binds DNA upstream and downstream of TATA box
-bridges TBP and RNAP II
-makes PIC asymmetric
-determines polarity
-N terminal inserts into Pol II RNA exit channel
-supports NTP binding for initiation |
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mcs5109 Wed, 19 Nov 2008 02:45:56 GMT |
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| TFIID |
-where histone-like TAFs are found |
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mcs5109 Wed, 19 Nov 2008 02:45:56 GMT |
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| TAFs |
-TBA associated factors
-TBA is associated w/ about 10 TAFs
-ex: Inr and DPEs
-similar to histone proteins
-histone-like TAFs found in TFIID complex and with histone modification enzymes |
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mcs5109 Wed, 19 Nov 2008 02:45:56 GMT |
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| SAGA |
histone modification enzyme |
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mcs5109 Wed, 19 Nov 2008 01:45:24 GMT |
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| TAFs (TBP-associated factors) |
-bind DNA elements at the promoter (Inr and DPE)
-Several TAFs have structural homology to histone; binds in similar manner?
-Histone like TAFs are found not only in the TFIID but are also associated with some histone modification enzymes (e.g., SAGA) |
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mcs5109 Wed, 19 Nov 2008 01:45:24 GMT |
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| TBP Binds to and Distorts DNA |
-TBP binds in the narrow groove of DNA at the TATA box
-many (but not all) genes transcribed by Pol II
-TBP bends the DNA about 80 degrees.
-TBP binds the DNA from minor groove.
-Uses a b Sheet Inserted into the Minor Groove |
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mcs5109 Wed, 19 Nov 2008 01:45:24 GMT |
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| euk GTFs |
-generally have increasing number of subunits |
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mcs5109 Wed, 19 Nov 2008 01:45:24 GMT |
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| RNAP II Initiation |
-euk promoter melting requires ATP, THIIH
-Pol II has CTD aka "tail"
-Pol II initiates transcription only when the CTD is largely unphosphorylated, but elongates only when phosphorylated
-CTD contains repeats of heptad Tyr-Ser-Pro-Thr-Ser-Pro-Ser
-52 repeats in mammal, 27 in yeast, other eukaryotes have intermediate values
-each repeat contains sites for phosphorylation by specific kinases (including one that is a subunit of TFIIH and P-TEFb).
-phosphorylated CTD provides the binding sites for numerous auxiliary factors (e.g., for 5’-mRNA capping, 3’ Poly A tail , Splicing and termination factors) |
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mcs5109 Wed, 19 Nov 2008 01:34:45 GMT |
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| When does Pol II initiate transcription? |
-when the CTD is largely unphosphorylated
-but, elongates only after the CTD has been phosphorylated |
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mcs5109 Wed, 19 Nov 2008 01:34:45 GMT |
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| RNAP II promoter melting |
-requires hydrolysis of ATP
-mediated by THIIH |
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mcs5109 Wed, 19 Nov 2008 01:34:45 GMT |
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| General transcription factors (GTIFs) |
-proteins required for specific transcription from a minimal promoter (core)
-not subunits of purified RNAPs
-required for RNAP to bind specifically to promoters.
-GTFs for Pol II are called TFIIx, where x = A, B, D, …
(no C)
Can have multiple subunits |
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mcs5109 Wed, 19 Nov 2008 01:34:45 GMT |
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| pre-initiation complex |
-complete set of general transcription factors and polymerase, bound together at the promoter and poised for initiation
-RNAP II |
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mcs5109 Wed, 19 Nov 2008 01:34:45 GMT |
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| in vivo transcription sites |
-upstream (in general) of the core promoter contains other elements
-regulatory sequence, enhancer
- can be located many 10s or even 100s of kb from the core promoters (both upstream and downstream) |
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mcs5109 Wed, 19 Nov 2008 01:04:21 GMT |
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| DCE and DPE |
-downstream promoter elements |
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mcs5109 Wed, 19 Nov 2008 01:02:45 GMT |
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| Inr |
initiator |
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mcs5109 Wed, 19 Nov 2008 01:02:45 GMT |
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| TATA box |
-TBP recognition element |
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mcs5109 Wed, 19 Nov 2008 01:02:45 GMT |
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| BRE |
-TFIIB recognition element |
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mcs5109 Wed, 19 Nov 2008 01:02:45 GMT |
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| RNAP II Core Promoters Are Made up of Combinations of Four Different Sequence Elements |
-core promoter= 40 nt long
-extends upstream or downstream of transcription start site
1. BRE
2. TATA box
3. Inr
4. downstream promoters DCE and DPE
-typical core promoter has only 2 or 3 of 4 elements
-in vivo, other elements required for transcription; can be located 100s kbases upstream and downstream from core promoter |
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mcs5109 Wed, 19 Nov 2008 01:02:45 GMT |
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| rut site |
-Rho utilization sites
-40 nt without secondary structures
-C rich
-remain largely single-stranded |
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mcs5109 Wed, 19 Nov 2008 00:46:09 GMT |
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| Rho |
-hexamer
-MW= 46 kDa
-RNA-dependent ATPase
-essential gene in E. coli
-recruited by rut site
-can't bind transcript being translated (ie ribosomes)
-only terminates transcripts still being transcribed beyond the end of a gene |
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mcs5109 Wed, 19 Nov 2008 00:46:09 GMT |
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| Rho-dependent termination |
-intrinsic terminator
-Rho binds to RNA and moves along it (tracks)
-when reaches a paused RNAP, causes it to dissociate and unwinds RNA/DNA duplex
-uses ATP hydrolysis
-terminates transcription |
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mcs5109 Wed, 19 Nov 2008 00:46:09 GMT |
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| How does hairpin disrupt elongation process? |
-steric clash: forcing open RNA exit channel
-modify active center conformation to disengage 3'-OH model from active center |
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mcs5109 Wed, 19 Nov 2008 00:36:54 GMT |
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| AU base pairs |
-weakest of all base pairs
-most easily disrupted by the effect of the stem loop in TEC |
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mcs5109 Wed, 19 Nov 2008 00:36:54 GMT |
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| Rho-independent termination |
-intrinsic terminator
-no other factors needed to work
-2 sequence elements:
1. short, inverted repeat of GCs upstream
2. followed by 4-10 AT's (A's on template)
-function in RNA
-inverted repeat forms hairpin in RNA
-disrupts elongation process
-U sequence downstream
-RNA only held to template at active site by A:U base pairings
-AU weakest of all
-RNA dissociates easily |
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mcs5109 Wed, 19 Nov 2008 00:36:54 GMT |
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| Box 12-2 The Single-Subunit RNA Polymerases |
-cellular organisms (bacteria, archaea and eukaryote) have multi-subunit RNAPs
Bacteriophage, chloroplast and mitochondria have single-subunit RNAP (T7-phage type single-subunit RNAP family)
MW: ~80 kDa, Right-hand structure. DNA pol I family.
Recognizes ds-DNA promoter (some species recognizes ss-DNA), unwinds ds-DNA for open complex formation and produces abortive products.
Chloroplast and mitochondria have also bacteria type multi-subunit RNAP. |
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mcs5109 Tue, 18 Nov 2008 08:52:02 GMT |
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| TFIIS |
-enhances hydrolytic editing in euk RNAP
-provide Mg++ at the active center of RNAP to carry out the reaction |
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mcs5109 Tue, 18 Nov 2008 08:50:20 GMT |
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| Gre |
-enhances hydrolytic editing in bacterial RNAP
-provide Mg++ at the active center of RNAP to carry out the reaction |
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mcs5109 Tue, 18 Nov 2008 08:50:20 GMT |
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| hydrolysis editing |
-RNAP backtracks by one or more nt
-cleaves the RNA product
-removes error-containing sequence
-stimulated by transcription elongation factors Gre (in bacteria) and TFIIS (in eukaryote)
-These factors provide Mg++ at the active center of RNAP to carry out the reaction |
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mcs5109 Tue, 18 Nov 2008 08:50:20 GMT |
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| pyrophosphorolytic editing |
-a simple back-reaction of RNA synthesis, requires pyrophosphate (PPi)
-removes 1 NTP by reincorporation of PPi |
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mcs5109 Tue, 18 Nov 2008 08:50:20 GMT |
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| 2 proofreading functions in RNAP |
1. pyrophosphorolytic editing
2. hydrolysis editing |
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mcs5109 Tue, 18 Nov 2008 08:50:20 GMT |
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| The Elongating Polymerase Is a Processive Machine that Synthesizes and Proofreads RNA |
-when RNAP finishes abortive transcription, it is released from the promoter
-elongation highly processive process
-dissociations of transcription factors required throughout transcription initiation to elongation
(bacteria, s dissociates from the core enzyme)
(Euk TFIID and TFIIA appear to stay behind at the promoter after Pol II and other factors leave the initiation complex)
Elongation RNAP has ~8 bp DNA/RNA hybrid in the active site (Fig. 12-10)
The 3’ end of RNA must contact the active site for polymerization (Fig. 12-10c)
RNAP synthesizes and proofreads RNA by using the same active site (Fig. 12-10d) |
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mcs5109 Tue, 18 Nov 2008 08:43:40 GMT |
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| Sigma subunit (region 3.2) in bacteria and general transcription factor TFIIB (B-finger) in euks |
could provide extra interactions with the initiating ribonucleotides at the active center to allow first phosphodiester bond formation |
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mcs5109 Tue, 18 Nov 2008 08:33:39 GMT |
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| global transcription regulation |
-[nucleotide] is able to regulate transcription |
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mcs5109 Tue, 18 Nov 2008 08:33:38 GMT |
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| RNAP can initiate transcription w/o a primer |
-RNAPs require higher [nucleotide] to initiate transcription
-[nucleotide] is able to regulate transcription (global transcription regulation)
-RNAPs have to have extra interactions with the initiating ribonucleotides at the active center to allow first phosphodiester bond formation
Sigma subunit (region 3.2) in bacteria and general transcription factor TFIIB (B-finger) in eukaryote could provide extra interactions |
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mcs5109 Tue, 18 Nov 2008 08:33:38 GMT |
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| close vs. open complex |
OCF: DNA downstream from -10 melts
a2: stabilizes single-stranded non-template DNA
active center: only reached by template DNA
Upstream DNA wraps around RNAP |
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mcs5109 Tue, 18 Nov 2008 08:23:52 GMT |
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| Open complex formation depends on what? |
-temp |
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mcs5109 Tue, 18 Nov 2008 08:23:52 GMT |
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| Transition to the Open Complex Involves Structural Changes in RNA Polymerase and in the Promoter DNA |
-see formula in notes
-All steps of the transcription initiation can be regulated to control the final RNA transcript level
Open complex formation is temperature-dependent |
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mcs5109 Tue, 18 Nov 2008 08:23:52 GMT |
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| 3 ways to start transcription in bacterial RNA polymerase |
transient excursions
inchworming
scrunching (believed to be accurate) |
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mcs5109 Tue, 18 Nov 2008 08:31:29 GMT |
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| Is UP element recognized by sigma subunit? |
No
-recognized by carboxy terminal domain of alpha subunit (alpha CTD) |
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mcs5109 Tue, 18 Nov 2008 08:07:58 GMT |
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| sigma subunit 4 |
-recognizes -35 element via alpha-turn-alpha helix motif (4.2) |
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mcs5109 Tue, 18 Nov 2008 08:07:58 GMT |
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| sigma subunit 3 |
-recognizes extended -10 element (3.0) |
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mcs5109 Tue, 18 Nov 2008 08:07:58 GMT |
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| sigma subunit 2 |
-recognizes -10 DNA element (2.4)
-DNA melting (2.3) |
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mcs5109 Tue, 18 Nov 2008 08:07:58 GMT |
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| Sigma factor |
-divided into four conserved regions (1 through 4) and each conserved region can be divided into sub-regions (2.1, 2.2, 2.3 and 2.4)
s2: -10 element recognition (region 2.4) and DNA melting (region 2.3)
s3: extended -10 element recognition (region 3.0).
s4: -35 element recognition by helix-turn-helix motif (region 4.2)
UP-element is not recognized by sigma subunit. It is recognized by carboxyl terminal domains of the a subunit (aCTD) |
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mcs5109 Tue, 18 Nov 2008 08:07:58 GMT |
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| UP element |
-increases polymerase binding by providing an additional specific interaction b/w enzyme and DNA
-found in strong promoters |
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mcs5109 Tue, 18 Nov 2008 07:58:36 GMT |
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| extended -10 sequence |
-standard -10 sequence w/ additional short sequence element at its upstream end
-extra contacts made
-only in absence of -35 region |
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mcs5109 Tue, 18 Nov 2008 07:58:36 GMT |
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| promoters recognized by sigma factors have consensus sequence |
-10 sequence: TATAAT
-35 sequence: TTGACA
separated by 17-19 nts
recognized by sigma subunit
deviation from sigma subunit will decrease level of transcription
-RNAP binds -60 and +20
-UP element and -10 extender help, too, to transcribe |
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mcs5109 Tue, 18 Nov 2008 07:58:36 GMT |
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| Archaeal RNAP inhibitors |
none ID'd yet |
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mcs5109 Tue, 18 Nov 2008 07:42:39 GMT |
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| TBP and TFB |
-euk transcription factors required by archaeal RNAP |
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mcs5109 Tue, 18 Nov 2008 07:42:39 GMT |
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| Archaeal RNAP |
-only one type of RNAP which synthesizes all classes of RNAs (mRNA, rRNA and tRNA)
-subunit composition is similar to euk RNAPs
-most similar to euk Pol II (based on sequence similarity)
-requires euk general transcription factors: TBP and TFB (TFIIB homologue)
Inhibitors: not identified yet
Bacterial-like transcription factors regulate gene expression |
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mcs5109 Tue, 18 Nov 2008 07:42:39 GMT |
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| alpha amanitin |
-Mitochondrial, chloroplast, archaea and prokaryotic RNAPs insensitive to a-Amanitin
-produced in the poisonous mushroom Amanita phalloides (death cap)
-unusual bicyclic octapeptide compound
Despite the amatoxins’ high toxicity (5~6mg / 40g fresh mushroom kills human adult), they act slowly
-Death occurs earlier than several days after mushroom ingestion. This, in part, reflects the slow turnover of eukaryotic mRNAs and proteins |
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mcs5109 Tue, 18 Nov 2008 07:42:39 GMT |
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| euk Pol III |
-makes tRNAs, 5S rRNA, some snRNAs
-less sensitive to a-amanitin |
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mcs5109 Tue, 18 Nov 2008 07:29:50 GMT |
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| euk Pol II |
-makes all mRNA, some snRNAs
-very sensitive to a-amanitin |
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mcs5109 Tue, 18 Nov 2008 07:29:50 GMT |
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| euk Pol I |
-makes large rRNA
-insensitive to a-amanitin |
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mcs5109 Tue, 18 Nov 2008 07:29:50 GMT |
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| MW for euk RNAP |
about 500 kDa |
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mcs5109 Tue, 18 Nov 2008 07:29:50 GMT |
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| 3 Types of euk RNAPs |
-each synthesize different classes of RNAs
-each RNAP has general transcription factors determining promoter specificity
-MW for each RNAP about 500 kDa |
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mcs5109 Tue, 18 Nov 2008 07:29:50 GMT |
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| rifamicin |
-anti-tuberculosis treatment
-blocks RNA extension |
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mcs5109 Tue, 18 Nov 2008 07:25:42 GMT |
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| active center |
-found at base of pincer in bacterial RNAP
-2-metal ion catalytic mechanism for NTP addition
-2 Mg+2 needed |
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mcs5109 Tue, 18 Nov 2008 07:24:01 GMT |
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| 2 pincers |
-B and B' subunits in bacteria
-27 Angstrom channel for dsDNA
-secondary channel for NTP entry to active center |
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mcs5109 Tue, 18 Nov 2008 07:24:01 GMT |
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| shape of each enzyme resembles |
...a crab claw
-2 "pincers" by B and B', 2 largest subunits in bacterial case
-active site found at base of pincers
-called "active cleft center" |
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mcs5109 Tue, 18 Nov 2008 07:24:01 GMT |
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| sigma subunit of bacterial RNAP |
-determines promoter specificity
-part of holoenzyme |
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mcs5109 Tue, 18 Nov 2008 07:24:01 GMT |
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| bacterial RNA holoenzyme |
-core plus sigma subunit
-start RNA synthesis specifically at a promoter
-sigma determines promoter specificity |
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mcs5109 Tue, 18 Nov 2008 07:24:01 GMT |
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| Bacterial RNAP core |
-A2BB'w catalyzes RNA chain synthesis
-core alone capable of RNA synthesis
-closely related to euk polymerases |
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mcs5109 Tue, 18 Nov 2008 07:24:01 GMT |
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| Bacterial RNAP |
-bacterial RNAP core enzyme alone capable of synthesizing RNA
-closely related to euk RNAPs
-structure of bacterial RNAP core enzyme similar to that of yeast RNAP
-only type of RNAP that synthesizes all 3 RNAs (m, t, r)
-core= 2ABB'w |
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mcs5109 Tue, 18 Nov 2008 07:24:01 GMT |
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| RPB6 |
-subunit shared amongst 3 euk RNAPs |
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mcs5109 Tue, 18 Nov 2008 07:24:01 GMT |
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| Table 12.1 |
-bacteria has single RNA polymerase
-euks have 3
-each line has homologous subunits listed
-All of bacterial RNAP subunits are conserved among all cellular organisms
-euk RNAPs share some subunits |
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mcs5109 Tue, 18 Nov 2008 07:24:01 GMT |
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| RNA Polymerases come in many different forms, but share many features |
-all RNAPs perform essentially same rxn in all cells, from bacteria to humans
-made up of multiple subunits |
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mcs5109 Tue, 18 Nov 2008 07:24:01 GMT |
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| promoter |
RNAP can start RNA synthesis at a site on the DNA template |
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mcs5109 Tue, 18 Nov 2008 06:39:30 GMT |
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| transcription basic info |
-catalyzes the synthesis of RNA directed by DNA (or RNA) as a template
-makes RNA chain with sequence complementary to template strand of DNA
-non-sense strand, bottom strand |
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mcs5109 Tue, 18 Nov 2008 06:39:30 GMT |
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| What's more accurate: replication or transcription? |
replication
-lack of extensive proofreading mechanisms (though it does have 2)
-1 mistake in 10,000 |
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mcs5109 Tue, 18 Nov 2008 06:39:30 GMT |
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| Can multiple RNA polymerases transcribe the same gene? |
Yes
-even multiple RNAPs can transcribe same gene at once
-cell can synthesize a large number of transcripts from a single gene |
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mcs5109 Tue, 18 Nov 2008 06:36:17 GMT |
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| Does RNA remain base paired to template DNA? |
No
-enzyme displaces the growing chain only a few nucleotides behind where each RNA is added |
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mcs5109 Tue, 18 Nov 2008 06:36:17 GMT |
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| Does RNA polymerase need a primer? |
No
-initiates transcription de novo |
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mcs5109 Tue, 18 Nov 2008 06:36:17 GMT |
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| Is transcription similar to DNA replication? |
Yes!
-both involve enzymes that synthesize a new strand of nucleic acid complementary to a DNA template strand
-new strand is RNA |
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mcs5109 Tue, 18 Nov 2008 06:36:17 GMT |
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