The RNA Polymerase II General Transcription Machinery Prof. Michael Hampsey

Transcription

1 The RNA Polymerase II General Transcription Machinery Michael Hampsey, PhD Robert Wood Johnson Medical School Piscataway, New Jersey 1 Central Dogma of Molecular Biology DNA Stores genetic information transcription Francis H. C. Crick RNA Protein translation RNA polymerase II (Pol II) transcribes class II genes Transfers genetic information Executes genetic information 2 Eukaryotic RNA Polymerases I, II & III Pol I rrna Pol II Ribosome DNA mrna Protein Pol III trna 3 The screen versions of these slides have full details of copyright and acknowledgements 1

2 Introduction to Transcription Human genome sequence: revealed 30,000 proteinencoding genes. 1. How are these genes expressed? 2. How are these genes regulated in response to physiological and developmental stimuli? 4 Fundamental Questions 1. How does Pol II identify a gene? 2. How does Pol II initiate transcription? 3. How is transcription regulated in response to physiological and developmental factors? 5 Pol II Machinery: Overview of Topics Core promoter Minimum DNA sequence essential for accurate transcription initiation by Pol II Enhancers and silencers DNA sequence elements that stimulate or repress transcription from a linked promoter General transcription factors proteins essential for accurate initiation by Pol II; bind the core promoter to generate a transcription preinitiation complex (PIC) Pol II structure and function Mechanism of transcription initiation 6 The screen versions of these slides have full details of copyright and acknowledgements 2

3 The RNA Polymerase Promoter I. Bacterial Pol Promoter s factor Pol II. Eukaryotic Pol II Promoter GTFs Pol II GTFs Pol II Machinery: Overview of Topics Core promoter Minimum DNA sequence essential for accurate transcription initiation by Pol II Enhancers and silencers DNA sequence elements that stimulate or repress transcription from a linked promoter General transcription factors required for binding of Pol II to core promoter Structure and function of Pol II Mechanism of transcription initiation 8 Pol II Core Promoter Elements BRE TFIIB Recognition Element TATA BOX Binds TBP INR Initiator DPE Downstream Core Promoter Element 9 The screen versions of these slides have full details of copyright and acknowledgements 3

4 CpG Islands as Proximal Promoter Elements kb regions with high density of CpG dinucleotides ~29,000 CpG island in human genome ~60% of human promoters consist of CpG islands, typically located within 150 bp of +1 Typically lack other core promoter elements Include binding sites for the transcription factor Sp1 Sp1 might recruit basal machinery to form the PIC Sp1 proteins PIC CpG Islands Summary: Core Promoter Elements Functionally defined as the minimum DNA sequence required for accurate transcription initiation by Pol II Essential for correct positioning and orientation of Pol II at the transcription start site Binds the GTFs Most promoters contain one or more of core elements No single element is essential for function Likely to be additional core elements: Gene-specific elements 11 Pol II Machinery: Overview of Topics Core promoter Minimum DNA sequence essential for accurate transcription initiation by Pol II Enhancers and silencers DNA sequence elements that stimulate or repress transcription from a linked promoter General transcription factors proteins essential for accurate initiation by Pol II; bind the core promoter to generate a transcription preinitiation complex (PIC) Pol II structure and function Mechanism of transcription initiation 12 The screen versions of these slides have full details of copyright and acknowledgements 4

5 Enhancer elements Activator Enhancer - 40 Core promoter + 40 Stimulate gene expression Binding sites for transcriptional activator proteins Located distal to the core promoter: sometimes hundreds or even thousand of bp away Typically located upstream of the core, but not necessarily so Can function in either orientation Counterpart in yeast: UAS (upstream activation sequence) 13 Enhanceosome A1 A2 A3 A4 E1 E2 E3 E4-40 Core promoter + 40 Multiple enhancers that act in combination to stimulate expression from a common promoter 14 UAS Elements (yeast) Activator UAS - 40 Core promoter + 40 Like enhancers, UAS (upstream activation sequences) are binding sites for transcriptional regulatory proteins In contrast to enhancers, UAS elements: are typically located within 500 bp of core promoter are always located upsteam of the core promoter and, orientation matters 15 The screen versions of these slides have full details of copyright and acknowledgements 5

6 Silencer elements Repressor Silencer - 40 Core promoter + 40 Binding sites for transcriptional repressor proteins Analogous to enhancers, with the opposite effect Mechanism of repression: through chromatin or through the core transcriptional machinery 16 URS Elements (yeast) Repressor URS - 40 Core promoter + 40 The yeast counterpart to metazoan silencers is the URS element Like silencers, URS elements are the binding site for regulatory proteins Repress transciption by affecting chromatin structure or Pol II recruitment. 17 Pol II Machinery: Overview of Topics Core promoter Minimum DNA sequence essential for accurate transcription initiation by Pol II Enhancers and silencers DNA sequence elements that stimulate or repress transcription from a linked promoter General transcription factors (GTFs) proteins essential for accurate initiation by Pol II; bind the core promoter to generate a transcription preinitiation complex (PIC) Pol II structure and function Mechanism of transcription initiation 18 The screen versions of these slides have full details of copyright and acknowledgements 6

7 Discovery of the GTFs Purified Pol II: catalytically active; but cannot initiate promoter-dependent transcription Pol II + human cell extract accurate initiation Provided an assay for the purification and identification of the general transcription factors. Accordingly, the GTFs, along w ith Pol II are both necessary and sufficient for accurate Pol II initiation in vitro 19 Discovery of the Pol II General Transcription Factors (GTFs) TFIIA TFIIB TFIID TBP Pol II Core Promoter TFIIE TFIIF TFIIH 20 The GTFs of RNA Polymerase II Required for accurate transcription initiation in vitro Yield low, basal level of transcription in vitro Remarkably, the same GTFs identified in human cells, were also found in rat, fly, yeast and other eukaryotes GTFs are universally required 21 The screen versions of these slides have full details of copyright and acknowledgements 7

8 Assembly of the Pol II Transcription Preinitiation Complex (PIC) PIC TFIIF TFIIA Pol II TFIIH TFIIB TFIID TBP TFIIE 22 The GTFs are loosely analogous to σ factor I. Bacterial RNAP Promoter s factor Pol II. Eukaryotic RNAPII Promoter Pol II GTFs GTFs TFIID TBP + 14 TBP-associated factors (TAFs) TAF4 TAF12 TBP TAF3 TAF10 TAF7 TAF8 TAF11 TAF14 TAF6 TAF5 TAF13 TAF9 TAF1 TAF2 Nucleates assembly of the transcription preinitiation complex 24 The screen versions of these slides have full details of copyright and acknowledgements 8

9 TBP Binds the TATA box Sits astride the TATA box as a molecular saddle Induces sharp bend in the DNA DNA-TBP complex: platform for assembly of TFIIA, TFIIB and the TAFs Universal transcription factor: plays critical role in transcription by Pol I, II and III TBP DNA-TBP 25 TAFs TBP-associated factors subunits include histone-fold motifs Form 5 histone-like pairs Initially described as requisite coactivators rather than GTFs TAFs are not universally required; promoters vary widely in their TAF requirement TAF-dependence defined by core promoter elements What do TAFs bind? 26 TAF Functions U A S Activator TAF4 TAF12 TBP TAF3 TAF10 TAF7 TAF8 TAF11 TAF14 TAF6 TAF5 TAF13 TAF9 TAF1 TAF TATA INR DPE 27 The screen versions of these slides have full details of copyright and acknowledgements 9

10 TFIID: 3D structure Electron microscopy: reveals TFIID to be a horseshoeshaped structure Grove could accommodate dsdna Two conformations: open and closed Suggests TFIID acts as molecular clamp to bind DNA 28 Class II promoters I. TAF-independent core promoters BRE TATA +1 II. TATA-less core promoters BRE INR DPE Is TFIID required? TBP-less TAF complexes TBP-less TAF complexes: SAGA (yeast) STAGA (human) TFTC (human) PCAF (human) None contains TBP; all contain a subset of TAFs Like TFIID, all include a histone acetyltransfer ase subunit TFIID: TAF1 Others: GCN5 Recognize sequence- specific elements of core promoters Recruited to promoter by activators Function as transcriptional coactivators 30 The screen versions of these slides have full details of copyright and acknowledgements 10

11 TLFs A host of TBP-like factors have been described: TLF TLP TRF2 TBP2 Metazoan, not yeast Function at TATA-less promoters Exert differential effects on developmental gene expression 31 TFIIA Stabilizes the TBP- DNA complex Blocks transcriptional inhibitors: Mot1 and NC2 Sometimes regarded as a cofactor rather than a GTF: not essential for accurate initiation TFIIA 32 TFIIB Binds the BRE element, TBP & Pol II Helps define the transcription start site. Regulates promoter clearance 3-D structures: Zn-ribbon B-finger Core domain ctfiib Courtesy ofstephan Burley, The Rockefeller University. 33 The screen versions of these slides have full details of copyright and acknowledgements 11

12 TFIIB: inserts into Pol II active center Mg 34 TFIIF TFIIF Dimer: RAP74 & RAP30 Binds Pol II Accompanies Pol II to the PIC Facilitates promoter opening (DNA melting) Like TFIIB, TFIIF affects start site selection 35 TFIIE TFIIE Heterodimer: 34 kda and 56 kda Binds the promoter near the transcription start site Creates docking site for TFIIH 3D structure: Like TFIIF, includes winged helix domains Counterpart to TFIIEα subunit (TFE) in Archaea 36 The screen versions of these slides have full details of copyright and acknowledgements 12

13 TFIIH Large multisubunit complex: - 10 subunits Five Catalytic Activities: Pol kda II CTD kinase Three (Cdk7-Cyclin Functions H or Kin28-Ccl1) Transcription: DNA-dependent promoter ATPase 5 melting to 3 DNA and Pol helicase II escape DNA repair 3 to 5 DNA helicase Cell E3 ubiquitin cycle progression ligase EM structure: cavity in center of complex suggests mechanism for promoter melting TFIIH 37 TFIIH 5-3 Helicase: Opens dsdna Tether XPB XPD p44 MAT1 p62 p52 p34 Cdk7 p8 Cyclin H 3-5 Helicase: Opens dsdna Ubiquitin ligase Phosphorylat es RNAP II CTD Ser5 Regulates Cdk7 activity 38 TFIIH: Clinical Importance Xeroderma pigmentosum (XP) trichothiodystrophy (TTD) Cockayne Syndrome (CS) 39 The screen versions of these slides have full details of copyright and acknowledgements 13

14 Fundamental Questions 1. How does Pol II identify a gene? 2. How does Pol II initiate transcription? 3. How is transcription regulated in response to physiological and developmental factors? 40 Pol II Machinery: Overview of Topics Core promoter Minimum DNA sequence essential for accurate transcription initiation by Pol II Enhancers and silencers DNA sequence elements that stimulate or repress transcription from a linked promoter General transcription factors (GTFs) proteins essential for accurate initiation by Pol II; bind the core promoter to generate a transcription preinitiation complex (PIC) Pol II structure and function Mechanism of transcription initiation 41 Pol II 12 subunit structure: Core domain: Rpb1, Rpb2, Rpb3, Rpb11 Subunits shared among RNAP I, II & III: Rpb5, Rpb6, Rpb8, Rpb10, Rpb12 Subunits not essential for elongation: Rpb4, Rpb7, Rpb9 42 The screen versions of these slides have full details of copyright and acknowledgements 14

15 Yeast Pol II 3D structure (10 subunit) core: Rpb3, 10, 11,12 + Rpb1,2 at active center jaws clamp RNA exit 43 Yeast RNAP II 12 subunit structure cleft clamp: closed conformation Rbp4/7 wedge core Rpb4 and Rpb7: Increases the surface area of the dock domain for PIC assembly Locks Pol II in closed conformation 44 "A Marvellous Machine for Making Messages" - A. Klug 45 The screen versions of these slides have full details of copyright and acknowledgements 15

16 The Pol II CTD Pol II (YSPTSPS) 26. CTD Variable number of repeats depending on organism complexity CTD coordinates RNA processing with transcription 5 -Cap addition Splicing 3 -end formation Nuclear export of mrna Platform for assembly of processing factors 46 Regulation of the Pol II CTD Undergoes cycles of phosphorylation/dephosphorylation Pol IIA unphosphorylated CTD Pol IIO phosphorylated CTD TFIIH phosphorylates serine-5 at transcription initiation Ctk1 complex phosphorylates serine-2 during elongation Ssu72 dephosphorylates serine-5-p Fcp1 dephosphorylates serine-2-p Ctk1 P P Cdk7/TFIIH Pol II (YSPTSPS) 26. CTD Fcp1 P Ssu72 P Pol II (YSPTSPS) 26. CTD 47 Pol II Machinery: Overview of Topics Core promoter Minimum DNA sequence essential for accurate transcription initiation by Pol II Enhancers and silencers DNA sequence elements that stimulate or repress transcription from a linked promoter General transcription factors (GTFs) proteins essential for accurate initiation by Pol II; bind the core promoter to generate a transcription preinitiation complex (PIC) Pol II structure and function Mechanism of transcription initiation 48 The screen versions of these slides have full details of copyright and acknowledgements 16

17 Pol II Transcription Cycle Pol II + GTFs Recruitment PIC Reinitiation + Pol II + TFIIB + TFIIF Isomerization Scaffold Complex - Pol II - TFIIB - TFIIF Open Complex NTPs Initiation Elongation Termination RNA 49 Scaffold: facilitates transcription reinitiaiton by Pol II Scaffold MEDIATOR TFIIF TFIIA Pol II TFIIH TFIIB -35TFIID TBP TFIIE 50 Fundamental Questions 1. How does Pol II identify a gene? 2. How does Pol II initiate transcription? 3. How is transcription regulated in response to physiological and developmental factors? 51 The screen versions of these slides have full details of copyright and acknowledgements 17

18 Activation by recruitment Activation domain Binding domain Enhancer SAGA MEDIATOR TFIID Pol II + GTFs Core promoter Fundamental Questions 1. How does Pol II identify a gene? 2. How does Pol II initiate transcription? 3. How is transcription regulated in response to physiological and developmental factors? 53 Transcriptional repression Repressor domain MEDIATOR Binding domain Silencer SSN6-TUP1 Core promoter The screen versions of these slides have full details of copyright and acknowledgements 18

19 Unanswered Questions How does Pol II recognize a promoter in the absence of defined core promoter elements? Proximal promoters Are there additional core promoter elements? Can core promoter elements be gene-specific? How do different combinations of core promoter elements contribute to regulation of gene expression? What is the mechanism of transcription reinitiation? How do activators facilitate PIC assembly? How do the GTFs interact to initiate transcription. What are the genetic switches that enable Pol II promoter escape? 55 The End MEDIATOR A1 A2 A3 A4 SAGA TFIID Pol II + GTFs E1 E2 E3 E4 DPE TATA INR DPE > enhanceosome core promoter The screen versions of these slides have full details of copyright and acknowledgements 19