A Movie of RNA Polymerase II Transcription
2012; Cell Press; Volume: 149; Issue: 7 Linguagem: Inglês
10.1016/j.cell.2012.06.006
ISSN1097-4172
AutoresAlan C. M. Cheung, Patrick Cramer,
Tópico(s)RNA Research and Splicing
ResumoWe provide here a molecular movie that captures key aspects of RNA polymerase II initiation and elongation. To create the movie, we combined structural snapshots of the initiation-elongation transition and of elongation, including nucleotide addition, translocation, pausing, proofreading, backtracking, arrest, reactivation, and inhibition. The movie reveals open questions about the mechanism of transcription and provides a useful teaching tool. We provide here a molecular movie that captures key aspects of RNA polymerase II initiation and elongation. To create the movie, we combined structural snapshots of the initiation-elongation transition and of elongation, including nucleotide addition, translocation, pausing, proofreading, backtracking, arrest, reactivation, and inhibition. The movie reveals open questions about the mechanism of transcription and provides a useful teaching tool. 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Cramer P. Structures of complete RNA polymerase II and its subcomplex, Rpb4/7.J. Biol. Chem. 2005; 280: 7131-7134Crossref PubMed Scopus (176) Google Scholar)10 subunit Pol IIS. cerevisiae1I50(Cramer et al., 2001Cramer P. Bushnell D.A. Kornberg R.D. Structural basis of transcription: RNA polymerase II at 2.8 angstrom resolution.Science. 2001; 292: 1863-1876Crossref PubMed Scopus (966) Google Scholar)12 subunit Pol IIS. cerevisiae1WCM(Armache et al., 2005Armache K.J. Mitterweger S. Meinhart A. Cramer P. Structures of complete RNA polymerase II and its subcomplex, Rpb4/7.J. Biol. Chem. 2005; 280: 7131-7134Crossref PubMed Scopus (176) Google Scholar)TFIIF dimerization moduleH. sapiens1F3UaHomology modeling was used to generate a model for S. cerevisiae.(Gaiser et al., 2000Gaiser F. Tan S. Richmond T.J. Novel dimerization fold of RAP30/RAP74 in human TFIIF at 1.7 A resolution.J. Mol. Biol. 2000; 302: 1119-1127Crossref PubMed Scopus (72) Google Scholar)Pol II-TFIIF complex modelS. cerevisiaeaHomology modeling was used to generate a model for S. cerevisiae., bModeled from crosslinking data.(Chen et al., 2010Chen Z.A. Jawhari A. Fischer L. Buchen C. Tahir S. Kamenski T. Rasmussen M. Lariviere L. Bukowski-Wills J.-C. Nilges M. et al.Architecture of the RNA polymerase II-TFIIF complex revealed by cross-linking and mass spectrometry.EMBO J. 2010; 29: 717-726Crossref PubMed Scopus (324) Google Scholar)TBP-TFIIB-DNA complexH. sapiens/A. thaliana1VOLcThese structures were combined to create open and closed promoter complex models (Kostrewa et al., 2009).(Nikolov et al., 1995Nikolov D.B. Chen H. Halay E.D. Usheva A.A. Hisatake K. Lee D.K. Roeder R.G. Burley S.K. Crystal structure of a TFIIB-TBP-TATA-element ternary complex.Nature. 1995; 377: 119-128Crossref PubMed Scopus (482) Google Scholar)Pol II-TFIIB complexS. cerevisiae3K1FcThese structures were combined to create open and closed promoter complex models (Kostrewa et al., 2009).(Kostrewa et al., 2009Kostrewa D. Zeller M.E. Armache K.-J. Seizl M. Leike K. Thomm M. Cramer P. RNA polymerase II-TFIIB structure and mechanism of transcription initiation.Nature. 2009; 462: 323-330Crossref PubMed Scopus (227) Google Scholar)Pol II partial open complex with DNAS. cerevisiae4A3I(Cheung et al., 2011Cheung A.C. Sainsbury S. Cramer P. Structural basis of initial RNA polymerase II transcription.EMBO J. 2011; 30: 4755-4763Crossref PubMed Scopus (68) Google Scholar)Minimal initially transcribing complexesS. cerevisiaedPDB codes 4A3G, 4A3J, 4A3B, 4A3M, 4A3C, 4A3E, 4A3D, 4A3F, 4A3K, and 4A3L.(Cheung et al., 2011Cheung A.C. Sainsbury S. Cramer P. Structural basis of initial RNA polymerase II transcription.EMBO J. 2011; 30: 4755-4763Crossref PubMed Scopus (68) Google Scholar)Archaeal RNA polymerase clamp-Spt4/5 complexP. furiosis3QQCeThese structures were used to model the Pol II-Spt4/5 EC (Martinez-Rucobo et al., 2011).(Martinez-Rucobo et al., 2011Martinez-Rucobo F.W. Sainsbury S. Cheung A.C.M. Cramer P. Architecture of the RNA polymerase-Spt4/5 complex and basis of universal transcription processivity.EMBO J. 2011; 30: 1302-1310Crossref PubMed Scopus (185) Google Scholar)Eukaryotic Spt4/5S. cerevisiae2EXUeThese structures were used to model the Pol II-Spt4/5 EC (Martinez-Rucobo et al., 2011).(Guo et al., 2008Guo M. Xu F. Yamada J. Egelhofer T. Gao Y. Hartzog G.A. Teng M. Niu L. Core structure of the yeast spt4-spt5 complex: a conserved module for regulation of transcription elongation.Structure. 2008; 16: 1649-1658Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar)Pol II EC with exiting RNA, upstream DNA duplex, and nontemplate strandS. cerevisiaeeThese structures were used to model the Pol II-Spt4/5 EC (Martinez-Rucobo et al., 2011)., fPositions of the RNA, upstream DNA duplex, and nontemplate strand were determined by using single-molecule FRET.(Andrecka et al., 2008Andrecka J. Lewis R. Brückner F. Lehmann E. Cramer P. Michaelis J. Single-molecule tracking of mRNA exiting from RNA polymerase II.Proc. Natl. Acad. Sci. USA. 2008; 105: 135-140Crossref PubMed Scopus (92) Google Scholar, Andrecka et al., 2009Andrecka J. Treutlein B. Arcusa M.A. Muschielok A. Lewis R. Cheung A.C.M. Cramer P. Michaelis J. Nano positioning system reveals the course of upstream and nontemplate DNA within the RNA polymerase II elongation complex.Nucleic Acids Res. 2009; 37: 5803-5809Crossref PubMed Scopus (77) Google Scholar)Posttranslocated complete ECS. cerevisiae1Y1WeThese structures were used to model the Pol II-Spt4/5 EC (Martinez-Rucobo et al., 2011)., gUsed to model the nucleotide addition cycle.(Kettenberger et al., 2004Kettenberger H. Armache K.J. Cramer P. Complete RNA polymerase II elongation complex structure and its interactions with NTP and TFIIS.Mol. Cell. 2004; 16: 955-965Abstract Full Text Full Text PDF PubMed Scopus (344) Google Scholar)Bacterial EC substrate complex, preinsertion stateT. thermophilus2PPBgUsed to model the nucleotide addition cycle.(Vassylyev et al., 2007bVassylyev D.G. Vassylyeva M.N. Zhang J. Palangat M. Artsimovitch I. Landick R. Structural basis for substrate loading in bacterial RNA polymerase.Nature. 2007; 448: 163-168Crossref PubMed Scopus (287) Google Scholar)EC substrate complex, insertion stateS. cerevisiae2E2HgUsed to model the nucleotide addition cycle.(Wang et al., 2006Wang D. Bushnell D.A. Westover K.D. Kaplan C.D. Kornberg R.D. Structural basis of transcription: role of the trigger loop in substrate specificity and catalysis.Cell. 2006; 127: 941-954Abstract Full Text Full Text PDF PubMed Scopus (361) Google Scholar)Pretranslocated ECS. cerevisiae1I6HgUsed to model the nucleotide addition cycle.(Gnatt et al., 2001Gnatt A.L. Cramer P. Fu J. Bushnell D.A. Kornberg R.D. Structural basis of transcription: an RNA polymerase II elongation complex at 3.3 A resolution.Science. 2001; 292: 1876-1882Crossref PubMed Scopus (744) Google Scholar)α-Amanitin-inhibited ECS. cerevisiae2VUMgUsed to model the nucleotide addition cycle.(Brueckner and Cramer, 2008Brueckner F. Cramer P. Structural basis of transcription inhibition by alpha-amanitin and implications for RNA polymerase II translocation.Nat. Struct. Mol. Biol. 2008; 15: 811-818Crossref PubMed Scopus (191) Google Scholar)Bacterial RNA polymerase ECT. thermophilus1IW7gUsed to model the nucleotide addition cycle.(Vassylyev et al., 2002Vassylyev D.G. Sekine S. Laptenko O. Lee J. Vassylyeva M.N. Borukhov S. Yokoyama S. Crystal structure of a bacterial RNA polymerase holoenzyme at 2.6 A resolution.Nature. 2002; 417: 712-719Crossref PubMed Scopus (627) Google Scholar)Paused Pol II with frayed RNAS. cerevisiae3HOU(Sydow et al., 2009Sydow J.F. Brueckner F. Cheung A.C.M. Damsma G.E. Dengl S. Lehmann E. Vassylyev D. Cramer P. Structural basis of transcription: mismatch-specific fidelity mechanisms and paused RNA polymerase II with frayed RNA.Mol. Cell. 2009; 34: 710-721Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar)Arrested Pol II with backtracked RNAS. cerevisiae3PO2(Cheung and Cramer, 2011Cheung A.C. Cramer P. Structural basis of RNA polymerase II backtracking, arrest and reactivation.Nature. 2011; 471: 249-253Crossref PubMed Scopus (243) Google Scholar)Pol II reactivation intermediate with TFIISS. cerevisiae3PO3(Cheung and Cramer, 2011Cheung A.C. Cramer P. Structural basis of RNA polymerase II backtracking, arrest and reactivation.Nature. 2011; 471: 249-253Crossref PubMed Scopus (243) Google Scholar)a Homology modeling was used to generate a model for S. cerevisiae.b Modeled from crosslinking data.c These structures were combined to create open and closed promoter complex models (Kostrewa et al., 2009Kostrewa D. Zeller M.E. Armache K.-J. Seizl M. Leike K. Thomm M. Cramer P. RNA polymerase II-TFIIB structure and mechanism of transcription initiation.Nature. 2009; 462: 323-330Crossref PubMed Scopus (227) Google Scholar).d PDB codes 4A3G, 4A3J, 4A3B, 4A3M, 4A3C, 4A3E, 4A3D, 4A3F, 4A3K, and 4A3L.e These structures were used to model the Pol II-Spt4/5 EC (Martinez-Rucobo et al., 2011Martinez-Rucobo F.W. Sainsbury S. Cheung A.C.M. Cramer P. Architecture of the RNA polymerase-Spt4/5 complex and basis of universal transcription processivity.EMBO J. 2011; 30: 1302-1310Crossref PubMed Scopus (185) Google Scholar).f Positions of the RNA, upstream DNA duplex, and nontemplate strand were determined by using single-molecule FRET.g Used to model the nucleotide addition cycle. Open table in a new tab Figure 2Snapshots from the MovieShow full captionRepresentative still images from the movie corresponding to the seven functional states shown in Figure 1 (A, initiation-competent complete Pol II-TFIIF complex; B, minimal closed promoter complex; C, minimal open promoter complex; D, initially transcribing complex; E, Pol II-Spt4/5 elongation complex; F, arrested complex; G, Pol II-TFIIS reactivation intermediate). Side views are depicted except for (A), which shows the front view (Cramer et al., 2001Cramer P. Bushnell D.A. Kornberg R.D. Structural basis of transcription: RNA polymerase II at 2.8 angstrom resolution.Science. 2001; 292: 1863-1876Crossref PubMed Scopus (966) Google Scholar).View Large Image Figure ViewerDownload Hi-res image Download (PPT) Representative still images from the movie corresponding to the seven functional states shown in Figure 1 (A, initiation-competent complete Pol II-TFIIF complex; B, minimal closed promoter complex; C, minimal open promoter complex; D, initially transcribing complex; E, Pol II-Spt4/5 elongation complex; F, arrested complex; G, Pol II-TFIIS reactivation intermediate). Side views are depicted except for (A), which shows the front view (Cramer et al., 2001Cramer P. Bushnell D.A. Kornberg R.D. Structural basis of transcription: RNA polymerase II at 2.8 angstrom resolution.Science. 2001; 292: 1863-1876Crossref PubMed Scopus (966) Google Scholar). All structures and models were superimposed onto the structure of the 12 subunit Pol II (PDB code 1WCM) (Armache et al., 2005Armache K.J. Mitterweger S. Meinhart A. Cramer P. Structures of complete RNA polymerase II and its subcomplex, Rpb4/7.J. Biol. Chem. 2005; 280: 7131-7134Crossref PubMed Scopus (176) Google Scholar), which was used as a reference. Modeling, prior to animation, was performed with Coot (Emsley et al., 2010Emsley P. Lohkamp B. Scott W.G. Cowtan K. Features and development of Coot.Acta Crystallogr. D Biol. Crystallogr. 2010; 66: 486-501Crossref PubMed Scopus (17085) Google Scholar). We used UCSF Chimera to generate all animations, labeling, and morphing interpolations between structures (Pettersen et al., 2004Pettersen E.F. Goddard T.D. Huang C.C. Couch G.S. Greenblatt D.M. Meng E.C. Ferrin T.E. UCSF Chimera—a visualization system for exploratory research and analysis.J. Comput. Chem. 2004; 25: 1605-1612Crossref PubMed Scopus (27950) Google Scholar), and then we used FFMPEG (http://ffmpeg.org) to encode the resulting image frames into video. Initiation and elongation factors are colored green and orange, respectively, and flexible polypeptide chains are represented by dotted lines. The orientations of Pol II complexes shown in the movie are restricted to either the front or side views used in earlier publications (Cramer et al., 2001Cramer P. Bushnell D.A. Kornberg R.D. Structural basis of transcription: RNA polymerase II at 2.8 angstrom resolution.Science. 2001; 292: 1863-1876Crossref PubMed Scopus (966) Google Scholar) (Figure 2). See Movie S1 online to download for teaching purposes. The movie starts with the formation of the initiation-competent Pol II-TFIIF complex (Figures 1A and 2A). Binding of the 10 subunit Pol II core (Cramer et al., 2001Cramer P. Bushnell D.A. Kornberg R.D. Structural basis of transcription: RNA polymerase II at 2.8 angstrom resolution.Science. 2001; 292: 1863-1876Crossref PubMed Scopus (966) Google Scholar) to the Pol II subcomplex Rpb4/7 (Armache et al., 2005Armache K.J. Mitterweger S. Meinhart A. Cramer P. Structures of complete RNA polymerase II and its subcomplex, Rpb4/7.J. Biol. Chem. 2005; 280: 7131-7134Crossref PubMed Scopus (176) Google Scholar) generates the complete, 12 subunit enzyme (Armache et al., 2003Armache K.J. Kettenberger H. Cramer P. Architecture of initiation-competent 12-subunit RNA polymerase II.Proc. Natl. Acad. Sci. USA. 2003; 100: 6964-6968Crossref PubMed Scopus (195) Google Scholar, Bushnell and Kornberg, 2003Bushnell D.A. Kornberg R.D. Complete, 12-subunit RNA polymerase II at 4.1-A resolution: implications for the initiation of transcription. Proc.Natl. Acad. Sci. USA. 2003; 100: 6969-6973Crossref PubMed Scopus (222) Google Scholar). Rpb4/7 binding stabilizes a closed conformation of the Pol II clamp domain, which only permits passage of single-stranded DNA to the active site (Armache et al., 2003Armache K.J. Kettenberger H. Cramer P. Architecture of initiation-competent 12-subunit RNA polymerase II.Proc. Natl. Acad. Sci. USA. 2003; 100: 6964-6968Crossref PubMed Scopus (195) Google Scholar). The complete Pol II is apparently relevant for initiation and elongation because Rpb4/7 is required for initiation in vitro (Edwards et al., 1991Edwards A.M. Kane C.M. Young R.A. Kornberg R.D. Two dissociable subunits of yeast RNA polymerase II stimulate the initiation of transcription at a promoter in vitro.J. Biol. Chem. 1991; 266: 71-75Abstract Full Text PDF PubMed Google Scholar) and because the complete Pol II is associated with the genome in vivo (Jasiak et al., 2008Jasiak A.J. Hartmann H. Karakasili E. Kalocsay M. Flatley A. Kremmer E. Strässer K. Martin D.E. Söding J. Cramer P. Genome-associated RNA polymerase II includes the dissociable Rpb4/7 subcomplex.J. Biol. Chem. 2008; 283: 26423-26427Crossref PubMed Scopus (39) Google Scholar). Subsequent binding of TFIIF to Pol II generates the complete Pol II-TFIIF complex. We positioned the dimerization domain of TFIIF on the lobe domain of Pol II based on crosslinking data (Chen et al., 2010Chen Z.A. Jawhari A. Fischer L. Buchen C. Tahir S. Kamenski T. Rasmussen M. Lariviere L. Bukowski-Wills J.-C. Nilges M. et al.Architecture of the RNA polymerase II-TFIIF complex revealed by cross-linking and mass spectrometry.EMBO J. 2010; 29: 717-726Crossref PubMed Scopus (324) Google Scholar, Eichner et al., 2010Eichner J. Chen H.-T. Warfield L. Hahn S. Position of the general transcription factor TFIIF within the RNA polymerase II transcription preinitiation complex.EMBO J. 2010; 29: 706-716Crossref PubMed Scopus (76) Google Scholar). In the next stage of the movie, the Pol II-TFIIF complex binds the TBP-TFIIB-DNA complex, resulting in a minimal closed promoter complex, in accordance with the classic model for initiation complex formation (Buratowski et al., 1989Buratowski S. Hahn S. Guarente L. Sharp P.A. Five intermediate complexes in transcription initiation by RNA polymerase II.Cell. 1989; 56: 549-561Abstract Full Text PDF PubMed Scopus (680) Google Scholar) (Figures 1B and 2B). The closed complex model was derived by combining crystal structures of the Pol II-TFIIB complex (Kostrewa et al., 2009Kostrewa D. Zeller M.E. Armache K.-J. Seizl M. Leike K. Thomm M. Cramer P. RNA polymerase II-TFIIB structure and mechanism of transcription initiation.Nature. 2009; 462: 323-330Crossref PubMed Scopus (227) Google Scholar, Liu et al., 2010Liu X. Bushnell D.A. Wang D. Calero G. Kornberg R.D. 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Evolution of multisubunit RNA polymerases in the three domains of l
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