week_9_lecture_1 - Required Reading for Week 9 Required...

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Unformatted text preview: Required Reading for Week 9: Required Weaver, Molecular Biology 5th Ed. Chapters 10-12 Chapters Week 9 Lecture 1 1st Hour: recap, quiz (15 min), eukaryotic RNA polymerases, structure of RNAP II and its elongation complex, RNAP II promoters Break 2nd Hour: RNAP II transcription factors, initiation, Mediator complex, elongation, enhancers and silencers Recap 1 Trp Operon Regulation by repression and Trp attenuation: attenuation: trpR_ - _trpO,P_____ trpL__ trpEDCBA__ - tryptophan: TrpR does not repress tryptophan: transcription; attenuator – ribosome stalling at TrpL (leader) trp codons blocks formation of two hairpin terminator terminator +tryptophan: TrpR represses transcription; +tryptophan: attenuator – no ribosome stalling at TrpL (leader) trp codons, formation of two hairpin terminator trp Recap 2 • Major shifts in bacterial transcription: – Gene cascades of phages - early (immediate Gene early) genes --> middle (delayed early) genes early) middle --> late genes late – Sigma factor switching (phage SPO1 of B. Sigma subtilis) subtilis) – T7: Class I (T7 RNAP) --> Class II --> Class III Class Class Class – T4: early (MotA/AsiA) --> middle --> late early middle (gp45 sliding clamp + gp55 s factor + gp33 coactivator) coactivator) Recap 3 Recap 3 Lambda regulatory control: lysis vs lysogeny IMMEDIATE EARLY: host RNAP makes N & cro from PL & PR DELAYED EARLY: N binds nut sites permits read­thru tL1 & tR1 and transcription extends into delayed early genes ­ cII, cIII, and Q LYSIS ­ or ­ LYSOGENY Q binds qut sites permits cII/cIII allows PRE cI mRNA, read­thru t & late genes transcribed cI binds OL & OR inhibits PL & PR but activates +ve autogenous cI expression from PRM -cI and cro, HTH proteins: helix3, recognition helix; helix2, phosphate backbone cro HTH binding binding -cI binds OR1 > OR2 > OR3… represses PR (& PL) transcription, induces PRM transcription --represses transcription, cI & LYSOGENY MAINTAINED cI positive control cI mutants defective for PRM transcription, RNAP suppressor mutations restore cI-dependent PRM transcription restore -cro binds OR3 > OR2 > OR1… represses PRM transcription and represses PR (& PL) represses transcription --- cI SYNTHESIS BLOCKED but Q permits late gene express & LYSIS transcription λ induction: SOS response/RecA stimulates cI autoproteolysis Also, see summary slides at the end of last lecture for lambda phage lytic/lysogenic Also, regulation recap regulation The regulatory elements in bacterial, yeast, and human genes EUKARYOTIC GENE EXPRESSION EUKARYOTIC • THREE EUKARYOTIC RNAPs: EUKARYOTIC RNAPs: >500kDa, 8-14 subunits Baker’s yeast (S. cerevisiae) RNAP II: genes RNAP encoding all subunits identified encoding core EUKARYOTIC RNAP: COEUKARYOTIC IMMUNOPRECIPITATON Weaver 2002 Molecular Biology Ed. 2 • Yeast RNAP II isolated by Yeast epitope tagging one subunit (Rpb3p) of the complex (Rpb3p) • All yeast proteins labeled All with radiolabel with • Antibody added and binds Antibody epitope - complex isolated by co-I.P. by • Bound proteins resolved on Bound gel & observed by autoradiography autoradiography EUKARYOTIC RNAP: SUBUNITS EUKARYOTIC Weaver 2002 Molecular Biology Ed. 2 • Epitope taggedRpb3p isolated in Rpb3p complex with other RNAP II subunits RNAP • Cells labeled with Cells [35S] to identify S] RNAP II proteins, and [32P]ATP to P]ATP identify phosphorylated subunits subunits • 12 subunits isolated 12 by multi-step purification (right) purification HUMAN AND YEAST RNA POLYMERASE II SUBUNITS core The crystal structure of yeast RNAP reveals a deep cleft that can accept a DNA template. The catalytic center with Mg2+ lies at the bottom of the cleft. A second Mg2+ comes in bound to the Incoming ribonucleotide The transcription bubble inside the RNAP II elongation complex If RNA-DNA hybrid were longer the rudder would be in the way RNA-DNA hybrid is 8 bp long ss downstream DNA facilitates turn Proposed translocation mechanism α- amanitin binds Yeast RNAP II • • • • The crystal structure of a transcription elongation complex involving yeast RNA pol II (lacking Rpb4/7) reveals that the clamp is closed over the RNA-DNA hybrid in the enzyme’s cleft, ensuring processivity of transcription. Three loops of the clamp – the rudder, lid, and zipper – appear to play important roles in, respectively: initiating dissociation of RNA-DNA hybrid, maintaining this dissociation, and maintaining dissociation of the template DNA. The active center of the enzyme lies at the end of pore 1, which appears to be the conduit for NTPs to enter the enzyme and for extruded RNA to exit the enzyme during backtracking A bridge helix lies adjacent to the active center, and flexing of this helix could play a role in translocation during transcription Kornberg and co-workers (2004) bound RNAP II to a set of synthetic oligos (partial ds DNA template and a 10 nt RNA product terminating in 3’-dA to trap polymerase in translocation state. Soaked crystals of this complex with correct or mismatched nucleotides striking differences revealed (A site and E site) Part of Rpb1 of Rpb1 Network of contacts with the GTP substrate in A site. The tripper loop of Rpb1 positions the substrate for incorporation and discriminates against improper nucleotides. of Rpb2 With the Rpb4/7 in place the clamp is forced shut. Because initiation occurs with the 12-subunit enzyme, with its clamp shut, it appears that the promoter DNA must melt before the template DNA strand can descend into the enzyme active site. It also appears that Rpb4/7 extends the dock region of the polymerase, making it easier for certain general transcription factors to bind, thereby facilitating transcription initiation OVERVIEW: RNAP II TRANSCRIPTION OVERVIEW: • • Unlike prokaryote transcription: (1) RNAP II requires general factors at Unlike general promoter for basal transcription; (2) promoters can be controlled by upstream factors, ubiquitous DNA-binding proteins that act on upstream ubiquitous promoters with consensus binding seq; (3) inducible factors activated at specific times or in specific tissues regulate promoters at response elements elements Eukaryote transcription unit: many regulatory seq found several kb upEukaryote or downstream; no defined terminators; initial transcript contains coding & noncoding regions EUKARYOTIC TRANSCRIPTION UNIT (RNAPII) EUKARYOTIC • PROMOTER: TATA box centered ~25bp upstream of PROMOTER: startpoint startpoint – Includes short elements (<10bp) that bind Includes transcription factors within ~200bp of start-site transcription – binds general transcription factors similar for all Pol II binds transcribed genes transcribed • ENHANCERS: elements up to several kb up/downstream ENHANCERS: -stimulate transcription initiation; bind specific transcription factors transcription MINIMUM REQUIREMENTS FOR RNAP II TRANSCRIPTION TRANSCRIPTION • • (i) Generic RNAP II promoter: shortest nt sequence that supports (i) RNAP II transcriptional initiation - basal transcription apparatus RNAP – Initiator (Inr): transcript start pt - no consensus seq; promoters with just Inr are recognized by RNAP II just Inr – many promoters have TATA box: ~25bp upstream from start site, only element with fixed position & found in all eukaryotes… – 8 bp consensus is all A-T base pairs and is similar to bacterial -10 bp sequence sequence – TATA-less promoters lack TATA box TATA (ii) Basal transcription apparatus: (ii) – RNAPII + general factors (TFIIs) constitute the basal apparatus complex – first step in complex formation is binding of TFIID to a region extending first TFIID upstream from TATA box upstream RECOGNITION: RNA POLYMERASE II CORE PROMOTER The eukaryotic core promoter refers to the minimal set of sequence elements for accurate transcription initiation by the Pol II machinery, as measured in vitro Typically ~40-60 nt long, extending either upstream or downstream from the +1 site TFIIB recognition (BRE), TATA box, the initiator (Inr), downstream promoter elements (DCEs, DPE, and MTE) Further upsteam: other regulatory elements, UASs, enhancers, silencers, boundary elements, and insulators HERPES VIRUS TK PROMOTER STUDIED WITH LINKER SCANNING MUTAGENESIS SCANNING Weaver 2002 Molecular Biology Ed. 2 HERPES VIRUS TK PROMOTER STUDIED WITH LINKER SCANNING MUTAGENESIS SCANNING RNA POLYMERASE I PROMOTER REGION: pol I is required for the expression of just one gene, rRNA precursor. There are many copies of that gene in each cell, and it is expressed at far higher levels than any other gene: so, has its own dedicated polymerase Upsteam control element where UBF binds and core (SL1 = TBP + 3TAFs specific for RNAP I transcription) Two rRNA promoter elements (RNAP I): the core element is absolutely required for any transcription to occur RNA POLYMERASE III (transcribes “short” genes) CORE PROMOTER Transcription initiation by RNA pol II (stepwise assembly) TATA is recognized by TFIID (multi-subunit complex: TBP+13TAFs, TBP-associated factors) TBP of TFIID binds TATA (TATA-binding protein) TFIID is critical factor in promoter recognition and preinitiation complex formation TBP extensively distorts the TATA sequence: provide a recruiting platform for other TFs PRIMER EXTENSION PRIMER • • • • • • • Weaver 2002 Molecular Biology Ed. 2 Primer extension: Primer (a) RNA isolated from sample (b) complementary labeled primer (b) added added (c) primer extended to 5´-end of (c) transcript with reverse transcriptase transcriptase (d) extended labeled ssDNA run (d) on gel (e) run sequencing rxn off (e) transcribed DNA using same primer primer (f) align fragment with sequence (f) to determine start site nucleotide to THIS TECHNIQUE MAY BE ALSO THIS USED TO QUANTITATE TRANSCRIPT LEVELS - THE MORE RNA, THE MORE REVERSE TRANSCRIPTS MADE TRANSCRIPTS TBP-DNA complex: TBP binds to and distorts DNA using a β sheet inserted into the minor groove A:T bases are favoured because they are more readily distorted to allow the initial opening of the minor groove There are also extensive interactions between the phosphate backbone and basic residues in the β sheet TBP: TATA-BINDING PROTEIN TBP: TBP • • • • Weaver 2002 Molecular Biology Ed. 2 TBP binds DNA minor groove forming “saddle” on DNA duplex ­ bends DNA (similar to CAP­cAMP) 80˚ at TATA box • TFIID consists of: TATAbinding protein (TBP) & 11 binding TBP-associated factors (TAFs) (TAFs) Different TAFs for different Different promoters promoters TFIID typically has a mass of TFIID ~800 kDa ~800 TBP “commitment factor” TBP binds RNAP II first & consigns promoter to be transcribed Bend in DNA caused by TBP Bend may serve to bring transcription factors and RNA polymerase closer together polymerase TBP/TATA BOX INTERACTIONS TBP/TATA huTBP huTBP produced in vaccina virus virus …or in or bacteria bacteria Weaver 2002 Molecular Biology Ed. 2 • Footprinting: TBP binds specifically to adenovirus major Footprinting: late TATA box (TBP is the only general transcription factor that makes sequence-specific DNA contacts) factor (b) no TBP binding to mutant TATA box (TTATCAT) (c) 180 a.a. C-terminal TBP region binds TATA box RNAP INITIATION COMPLEX RNAP • RNAP II initiation requires general RNAP factor complex assembly in defined order: order: • TFIID assembly (TBP and TAFs) ---> TFIID TFIIA ---> TFIIB ---> TFIIF TFIIA (2 subunits) ---> RNAP II ---> (DNA melting) ---> TFIIE ---> TFIIH & (J) ---> (promoter TFIIE clearance) clearance) Footprinting shows increased DNA Footprinting length covered as each subunit added: TFIIA -increased protection upstream TFIIB -protection downstream of TATA -protection box box • • • TAFs bind to DNA elements at the promoter, some have structural homology to histones TFIIB (one protein): crystal structure shows specific base contacts with the major groove upstream of BRE and the minor groove downstream of TATA Asymmetric binding of TFIIB to the TBP-TATA complex accounts for the asymmetry in the rest of assembly in the preinitiation complex and the unidirectional transcription TFIIB-TBP-promoter TFIIB-TBP-DNA TERNARY COMPLEX TFIIB-TBP-DNA • TFIIB binds TFIIB adjacent to TBP adjacent • TFIIB extends TFIIB contacts along one DNA face that provides surface for RNAP II recognition after TFIIF binding & RNAP recruitment RNAP Weaver 2002 Weaver Molecular Biology Ed. 2 Biology TFIIF TFIIF • The 2 TFIIF subunits are next to join complex The after TFIIB: after -larger TFIIF subunit, RAP74: ATP-dependent -larger DNA helicase that could be involved in melting duplex DNA at initiation melting -smaller subunit, RAP38: homology to -smaller bacterial σ factor regions that contact core polymerase - RAP38 binds tightly to RNAP II polymerase • TFIIF brings RNAP into assembling complex, TFIIF where CTD tail of polymerase interacts with TFIID. As with prokaryotes we get local DNA melting and abortive transcripts and DABPolF COMPLEX DABPolF Weaver 2002 Molecular Biology Ed. 2 • DNase footprinting of promoter DNA DNase with TFIID, A, B, and F, and RNAP II bound increases DNA protection by transcription complex to extend further downstream (to +17) further • Corresponds to RNAP II large size • TFIIF recruits RNAP II Weaver 2002 Molecular Biology Ed. 2 GENERAL TRANSCRIPTION FACTORS (GTFs): GENERAL PROMOTER CLEARANCE & ELONGATION Weaver 2002 Molecular Biology Ed. 2 • Promoter clearance: TFIIE & H required; TFIIE binding extends Promoter protected region to +30 ---> recruits TFIIH protected • TFIIH activities: ATPase, kinase, & also helicase that unwinds TFIIH even more Inr DNA allowing RNAP movement and promoter clearance clearance TFIIH promoter melting TFIIH • • • How does TFIIH mediate promoter How melting? It is now believed that a subunit of TFIIH It acts as an ATP-driven translocator of the double-stranded DNA. This subunit binds to the DNA downstream from polymerase, and feeds double-stranded DNA, with a right-handed threading, into the cleft of the polymerase the This action drives the melting of the This DNA because the upstream promoter DNA is held in fixed position by TFIID and the rest of GTFs and RECALL XP - XERODERMA PIGMENTOSUM • HUMAN EXCISION HUMAN REPAIR REPAIR • XPC-hHR23B XPC-hHR23B complex recognizes complex damage, binds & damage binds melts DNA melts • TFIIH helicase melts TFIIH more more • Endonucleases Endonucleases directed by RPA to RPA cut out damaged strand strand • DNA pol fills, ligase DNA seals seals Weaver 2002 Molecular Biology Ed. 2 TFIIH PHOSPHORYLATION OF RNAP II CTD TFIIH P labelled ATP added added 32 Weaver 2002 Molecular Biology Ed. 2 • TFIIH kinase phosphorylates CTD RNAP II tail TFIIH unphosphorylated preinitiation form (Pol IIA) ---> phosphorylated elongation form (Pol IIO) phosphorylated [Pol IIB lacks CTD and cannot be phosphorylated] • CTD tail alone (freed from RNAP with chymotrypsin [+Chym]) CTD phosphorylated by addition of TFIIH (+H) phosphorylated In vivo, transcription initiation requires additional proteins, including the Mediator complex A new set of factors stimulates RNA pol II elongation and RNA proofreading TFIIH P-TEFb Various proteins are through to stimulate elongation by pol II. One of these, kinase P-TEFb, is recruited to polymerase by transcriptional activators and, once bound, phosphorylates the serine residue at position 2 of the CTD repeats. Another factor, SPT5 (a universally conserved transcription factor across all three kingdoms of life), displaces initiation factors. Important because some promoters in higher eukaryotes recruit initiation complex effectively, but polymerase remains paused just after initiating transcription. Such promoters seem to be associated with genes that are poised to be expressed rapidly or in a highly coordinated fashion, and their expression is regulated through the recruitment of specific activators of the P-TEFb kinase, which releases them from their pause. β -GLOBIN PROMOTER: MUTAGENESIS REVEALS -GLOBIN UPSTREAM ELEMENTS UPSTREAM GC box GC (-90) CAAT box CAAT (approx -75) (approx TAT A box • Nucleotide changes introduced at every position 100bp Nucleotide upstream of β -GLOBIN start - most caused down mutations & clustered in 3 short regions: TATA box; CAAT TATA CAAT box (also associated with up mutations); GC box box GC (GGGCGG consensus) (GGGCGG β -GLOBIN PROMOTER: MUTAGENESIS REVEALS -GLOBIN UPSTREAM ELEMENTS UPSTREAM GC box GC (-90) CAAT box CAAT (approx -75) (approx TAT A box • 2 upstream elements are actually more effective upstream more components of the promoter than TATA components • CAAT box: one of first common upstream elements CAAT described, often at around -80 but can function at range of distances from the start site distances β -GLOBIN PROMOTER: MUTAGENESIS REVEALS -GLOBIN UPSTREAM ELEMENTS UPSTREAM GC box GC (-90) CAAT box CAAT (approx -75) (approx TAT A box • GC box is another common promoter element that often GC appears in multiple copies in a promoter, present in either orientation orientation • another common upstream element is octamer, which has another octamer which 8 bp consensus sequence bp TBP & TATA- or TATA-LESS PROMOTERS TBP • TATA-containing TATA-containing promoter: TBP binds promoter TATA box TATA • TATA-less TATA-less promoter: TBP cannot bind but tethered to TAFs bound at Inr bound • TATA-less with TATA-less GC boxes: TBP indirectly tethered by Sp1 bound to GC boxes boxes ELEMENT COMBINATIONS IN PROMOTERS ELEMENT • GC, CAAT, TATA GC, boxes & octamer sites (8bp consensus element) organized “mix & match” in promoters - no one element is common to all promoters all • Importance of Importance elements determined by base substitutions, deletion, linker scanning, and primer extension extension GC-BOXES IN SV40 EARLY PROMOTER STUDIED WITH BASE CHANGES BASE • Sp1 binds GC-boxes - transcription levels Sp1 reduced as no. of intact GC boxes declines reduced UPSTREAM TRANSCRIPTION FACTORS UPSTREAM • Module Consensus DNA DNA • TATA BOX TATAAA ~10bp • CAAT BOX GGCCAATCT ~22bp • GC BOX GGGCGG ~20bp • Octamer ATTTGCAT ~20bp • Octamer ATTTGCAT ~23bp • ΚB GGGACTTTCC ~10bp ~10bp • ATF GTGACGT ~20bp Factor TBP CTF/NF1 SP1 Oct-1 Oct-2 N FΚ B SP1 -Upstream elements are bound by factors that are generally ubiquitous, so Upstream most promoters which contain elements will be bound most -However, some upstream elements bound by several different factors ie. -However, ie CAAT box bound by tissue-specific factors CAAT -Footprinting indicates factors bind more than just consensus DNA-so -Footprinting upstream region could be covered with factors upstream UPSTREAM TRANSCRIPTION FACTORS UPSTREAM -TATA & Inr determine -TATA location of both basal complex assembly & transcript initiation transcript -Upstream elements affect -Upstream frequency of initiation, and therefore gene expression, therefore most likely by acting on the most TFIIs (i.e. general transcription factors) transcription - e.g. Sp1 binds to GC boxes e.g. and can be used to assemble TFIID at a TATA-less promoter through interaction with a TAF with ENHANCERS SV40 promoter • • • • • in addition to upstream elements found in promoter (found within a in few 100 bp of start site) other sequences called enhancers can enhancers increase promoter activity increase Enhancers: orientation & position independent; therefore, distinct Enhancers: from promoter from e.g. SV40 enhancer: ~200bp upstream, 2 X 72bp sequences required e.g. for high level transcription (many binding sites) for Mutational analysis of SV40 enhancer uncovered high density of Mutational elements (more so than β -globin promoter) Enhancers often contain same elements as found in promoters, (e.g. Enhancers octamer) octamer) IG γ 2b H-CHAIN ENHANCER Protein gel Northern Weaver 2002 Molecular Biology Ed. 2 X: cutting sites for XbaI No No expression expression • Enhancers can Enhancers confer tissueconfer specific specific transcription transcription...
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