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Berkeley MCELLBI 110 - Complex Transcription Machinery

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Complex TranscriptionMachinerySubunits of the Basal Txn ApparatusStepwise Assembly of the Pre-initiationComplexInterplay of Activators, Co-regulators andRNA Polymerase at the PromoterDivide and conquer: biochemical identification of generaltranscription factors (GTFs) required for accurate initiationHuman cell nuclear extractGTFs + RNA pol II + Cloned promoter DNASpecifically-initiatedmRNATFIIA B C D E…etcPhosphocelluloseBasal (‘General’) Transcription Factors for RNA Polymerase IISequential assembly ofthe pre-initiationcomplex (PIC) andopen complex by RNApolII + GTFsTFIID = TAFIIs +TFIID = TBP + ~12 TAFIIsTFIIB 1 subunitTFIIF 2 subunitsRNA pol II 12 subunitsTFIIE 2 subunitsTFIIH 12 subunits-Has helicase and kinase activityNon-phosphorylatedClosed complexTBP (TATA-binding protein)•Conserved C-terminal domain•Dyad symmetry•Binds multiple transcription proteins•Binds in the minor groove and significantly bends DNADISCOVERY OF TRANSCRIPTION CO-ACTIVATORS : TAF Subunits of TFIIDThree-dimensional structure of thehuman TFIID-IIA-IIB complex. Position of IIB and IIA on the TFIID structure and mappingof the TBP. The blue mesh corresponds to the holo-TFIID,with the A, B, and C lobes indicated. (A) The green meshcorresponds to the density difference between the holo-TFIIDand the TFIID-IIB complex. (B) The magenta and greenmeshes show the density difference between the holo-TFIIDand the trimeric complex TFIID-IIA-IIB. The densitydepicted in light green can be attributed to TFIIB bycomparison with (A), and the magenta density thereforecorresponds to IIA. (C) The yellow mesh shows the densitydifference between the holo-TFIID and TFIID that is boundto the TBP antibody.Tissue and Gene-Specific Core PromoterRecognition ComplexesUbiquitiousTissue-selectiveGene-specificModel for cooperative assembly of an activated transcription-initiation complex.Four activators enriched inhepatocytes plus the ubiquitousAP1 factor bind to sites in thehepatocytespecific enhancer andpromoter-proximal region of theTTR gene.The activation domains of thebound activators interactextensively with co-activators,TAF subunits of TFIID,Srb/Mediator proteins, andgeneral transcription factors,resulting in looping of the DNAand formation of a stableactivated initiation complex.Activators work in part by recruiting components of thetranscription machinery.Combinatorial possibilities due to formation of heterodimerictranscription factors. (a) In the hypothetical example shown,transcription factors A, B, and C can eachinteract with each other, permitting thethree factors to bind to six different DNAsequences (sites 1–6) and creating sixcombinations of activation domains. (Notethat each binding site is divided into twohalf-sites, and that a single heterodimericfactor contains the activation domains ofeach of its constituent monomers.)(b) When an inhibitory factor (green) isexpressed that interacts only with factor A,binding to sites 1, 4, and 5 is inhibited, butbinding to sites 2, 3, and 6 is unaffected.Inhibition by steric mechanismsIn the three mechanisms shown, therepressor either inhibits activation ordirectly interferes with formation of theinitiation complex. In addition, somerepressors interact with “co-repressor”proteins, that are thought to interact inturn with general transcription factors toinhibit initiation.Later, in the chromatin section, we willconsider another repression mechanisminvolving histone modification.Inhibitory regulation by truncated HLH proteins.The HLH motif is responsible for both dimerization and DNA binding. On the left, an HLHhomodimer recognizes a symmetric DNA sequence. On the right, the binding of a full-lengthHLH protein to a truncated HLH protein that lacks the DNA-binding helix generates aheterodimer that is unable to bind DNA tightly. If present in excess, the truncated proteinmolecule blocks the homodimerization of the full-length HLH protein and thereby prevents itfrom binding to DNA thus behaving as a dominant negative regulator.Major Points1. Fractionation, identification and purification of the basal trxn machinery: TFIIA,B,D,E,F, H and RNA Pol II2. Stepwise assembly of the pre-initiation complex (PIC) at RNA pol II promoters3. TBP , a subunit of TFIID, binds core promoter and bends DNA4. TFIID is composed of TBP and TAFs which bind promoter DNA and serves as a Co-activator to help link activators to the PIC5. Metazoans evolved the use of TFIID and Co-regulators that contain cell-type or tissue specific subunits6. Activators can induce large conformational changes in the structure of Co-activators7. Combinatorial regulation mediated by: heteromeric activators, activator/repressor interactions and dominant negative molecules8. Eukaryotes use a plethora of co-regulators (mediators, co-activators) to direct RNA pol II


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Berkeley MCELLBI 110 - Complex Transcription Machinery

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