Portal de Periódicos da CAPES

Evolution of Two Modes of Intrinsic RNA Polymerase Transcript Cleavage

2011; Elsevier BV; Volume: 286; Issue: 21 Linguagem: Inglês

10.1074/jbc.m111.222273

1083-351X

Wenjie Ruan, Elisabeth Lehmann, Michael Thomm, Dirk Kostrewa, Patrick Cramer,

RNA modifications and cancer

During gene transcription, the RNA polymerase (Pol) active center can catalyze RNA cleavage. This intrinsic cleavage activity is strong for Pol I and Pol III but very weak for Pol II. The reason for this difference is unclear because the active centers of the polymerases are virtually identical. Here we show that Pol II gains strong cleavage activity when the C-terminal zinc ribbon domain (C-ribbon) of subunit Rpb9 is replaced by its counterpart from the Pol III subunit C11. X-ray analysis shows that the C-ribbon has detached from its site on the Pol II surface and is mobile. Mutagenesis indicates that the C-ribbon transiently inserts into the Pol II pore to complement the active center. This mechanism is also used by transcription factor IIS, a factor that can bind Pol II and induce strong RNA cleavage. Together with published data, our results indicate that Pol I and Pol III contain catalytic C-ribbons that complement the active center, whereas Pol II contains a non-catalytic C-ribbon that is immobilized on the enzyme surface. Evolution of the Pol II system may have rendered mRNA transcript cleavage controllable by the dissociable factor transcription factor IIS to enable promoter-proximal gene regulation and elaborate 3′-processing and transcription termination. During gene transcription, the RNA polymerase (Pol) active center can catalyze RNA cleavage. This intrinsic cleavage activity is strong for Pol I and Pol III but very weak for Pol II. The reason for this difference is unclear because the active centers of the polymerases are virtually identical. Here we show that Pol II gains strong cleavage activity when the C-terminal zinc ribbon domain (C-ribbon) of subunit Rpb9 is replaced by its counterpart from the Pol III subunit C11. X-ray analysis shows that the C-ribbon has detached from its site on the Pol II surface and is mobile. Mutagenesis indicates that the C-ribbon transiently inserts into the Pol II pore to complement the active center. This mechanism is also used by transcription factor IIS, a factor that can bind Pol II and induce strong RNA cleavage. Together with published data, our results indicate that Pol I and Pol III contain catalytic C-ribbons that complement the active center, whereas Pol II contains a non-catalytic C-ribbon that is immobilized on the enzyme surface. Evolution of the Pol II system may have rendered mRNA transcript cleavage controllable by the dissociable factor transcription factor IIS to enable promoter-proximal gene regulation and elaborate 3′-processing and transcription termination. IntroductionThe eukaryotic RNA polymerases Pol 3The abbreviations used are: Pol, RNA polymerase; TFS, transcription factor S; TFIIS, transcription factor IIS. I, II, and III share a highly conserved active center (1Cramer P. Bushnell D.A. Kornberg R.D. Science. 2001; 292: 1863-1876Crossref PubMed Scopus (955) Google Scholar, 2Jasiak A.J. Armache K.J. Martens B. Jansen R.P. Cramer P. Mol. Cell. 2006; 23: 71-81Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar, 3Kuhn C.D. Geiger S.R. Baumli S. Gartmann M. Gerber J. Jennebach S. Mielke T. Tschochner H. Beckmann R. Cramer P. Cell. 2007; 131: 1260-1272Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar) that catalyzes DNA-dependent RNA synthesis during gene transcription. The same active center also catalyzes cleavage of the nascent transcript during proofreading and recovery from transcription arrest (4Sosunov V. Sosunova E. Mustaev A. Bass I. Nikiforov V. Goldfarb A. EMBO J. 2003; 22: 2234-2244Crossref PubMed Scopus (161) Google Scholar, 5Kettenberger H. Armache K.J. Cramer P. Cell. 2003; 114: 347-357Abstract Full Text Full Text PDF PubMed Scopus (264) Google Scholar). Transcript cleavage is essential for cell viability (6Sigurdsson S. Dirac-Svejstrup A.B. Svejstrup J.Q. Mol. Cell. 2010; 38: 202-210Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). Despite the active center conservation, the strength of this intrinsic cleavage activity greatly differs between polymerases. Although the cleavage activity is very strong for Pol I (3Kuhn C.D. Geiger S.R. Baumli S. Gartmann M. Gerber J. Jennebach S. Mielke T. Tschochner H. Beckmann R. Cramer P. Cell. 2007; 131: 1260-1272Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar) and Pol III (7Alic N. Ayoub N. Landrieux E. Favry E. Baudouin-Cornu P. Riva M. Carles C. Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 10400-10405Crossref PubMed Scopus (42) Google Scholar, 8Thuillier V. Brun I. Sentenac A. Werner M. EMBO J. 1996; 15: 618-629Crossref PubMed Scopus (37) Google Scholar), it is very weak for Pol II. The molecular basis for this phenomenon remains unknown.Intrinsic transcript cleavage requires the homologous subunits A12.2, Rpb9, and C11 in Pol I, II, and III, respectively (3Kuhn C.D. Geiger S.R. Baumli S. Gartmann M. Gerber J. Jennebach S. Mielke T. Tschochner H. Beckmann R. Cramer P. Cell. 2007; 131: 1260-1272Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar, 9Walmacq C. Kireeva M.L. Irvin J. Nedialkov Y. Lubkowska L. Malagon F. Strathern J.N. Kashlev M. J. Biol. Chem. 2009; 284: 19601-19612Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, 10Chédin S. Riva M. Schultz P. Sentenac A. Carles C. Genes Dev. 1998; 12: 3857-3871Crossref PubMed Scopus (151) Google Scholar). Archaea contain a related protein, TFS, which is required for RNA cleavage by the polymerase (11Hausner W. Lange U. Musfeldt M. J. Biol. Chem. 2000; 275: 12393-12399Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar, 12Lange U. Hausner W. Mol. Microbiol. 2004; 52: 1133-1143Crossref PubMed Scopus (48) Google Scholar). All these proteins consist of two zinc-binding β-ribbon domains. Rpb9 resides on the enzyme surface, where its N-terminal zinc ribbon (N-ribbon) forms part of the Rpb1/9 jaw, and its C-terminal zinc ribbon (C-ribbon) binds between the Rpb1 funnel domain and the Rpb2 domains lobe and external-1 (Fig. 1). The very weak cleavage activity of Pol II is greatly stimulated by TFIIS, which contains a Pol II-binding domain that is located at the Rpb1/9 jaw and a C-ribbon (5Kettenberger H. Armache K.J. Cramer P. Cell. 2003; 114: 347-357Abstract Full Text Full Text PDF PubMed Scopus (264) Google Scholar, 13Qian X. Jeon C. Yoon H. Agarwal K. Weiss M.A. Nature. 1993; 365: 277-279Crossref PubMed Scopus (111) Google Scholar). The TFIIS C-ribbon binds the pore beneath the Pol II active center and reaches the active site with a hairpin containing the invariant residues Arg-287, Asp-290, and Glu-291 that are required for function (5Kettenberger H. Armache K.J. Cramer P. Cell. 2003; 114: 347-357Abstract Full Text Full Text PDF PubMed Scopus (264) Google Scholar, 14Jeon C. Yoon H. Agarwal K. Proc. Natl. Acad. Sci. U.S.A. 1994; 91: 9106-9110Crossref PubMed Scopus (71) Google Scholar, 15Awrey D.E. Shimasaki N. Koth C. Weilbaecher R. Olmsted V. Kazanis S. Shan X. Arellano J. Arrowsmith C.H. Kane C.M. Edwards A.M. J. Biol. Chem. 1998; 273: 22595-22605Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar, 16Kettenberger H. Armache K.J. Cramer P. Mol. Cell. 2004; 16: 955-965Abstract Full Text Full Text PDF PubMed Scopus (344) Google Scholar, 17Wang D. Bushnell D.A. Huang X. Westover K.D. Levitt M. Kornberg R.D. Science. 2009; 324: 1203-1206Crossref PubMed Scopus (183) Google Scholar, 18Cheung A.C. Cramer P. Nature. 2011; 471: 249-253Crossref PubMed Scopus (239) Google Scholar). Although A12.2 and C11 contain these three hairpin residues, Rpb9 lacks the residue corresponding to Glu-291. It remains unclear how the ribbon domains are related evolutionarily and mechanistically and how this may result in different cleavage activities. Here we used a combination of mutagenesis, cleavage assays, and x-ray crystallography to unravel the molecular basis for differential intrinsic RNA cleavage activities of Pol II and Pol III and suggest how the C-ribbon domains are related evolutionarily and how different cleavage activities arose during evolution.DISCUSSIONOur results unravel the molecular basis for the difference in RNA cleavage activities of Pol II and Pol III. We show that replacement of the Rpb9 C-ribbon by the C11 C-ribbon confers strong intrinsic cleavage to Pol II. This unexpected gain of function stems from a switch in the cleavage mechanism, as suggested by x-ray crystallography and mutagenesis. Although the Rpb9 C-ribbon acts allosterically from the polymerase surface, the C11 C-ribbon acts directly by binding the pore and complementing the active center with its catalytic hairpin. Thus two modes exist for polymerase-intrinsic RNA cleavage, an allosteric, weak mode used by Rpb9, and a direct, strong mode used by C11 and TFIIS.Our results also suggest a model for how polymerase cleavage activities evolved (Fig. 1B). Pol I and Pol III have strong intrinsic cleavage activities because they contain homologues of archaeal TFS (A12.2 and C11, respectively) that contain C-ribbons with catalytic hairpins that can enter the pore to directly stimulate cleavage. In the Pol II system, the two domains are, however, part of two different polypeptides. Although the N-ribbon is part of Rpb9, the C-ribbon is part of TFIIS. During evolution, the C-ribbon likely duplicated and was altered in Rpb9 to attach the domain to the surface and to allow only for weak, allosteric cleavage induction.Our results are consistent with published data. First, mutation of the C11 hairpin residues is lethal (10Chédin S. Riva M. Schultz P. Sentenac A. Carles C. Genes Dev. 1998; 12: 3857-3871Crossref PubMed Scopus (151) Google Scholar). Second, the C11 C-ribbon is not observed on the surface in a recent electron microscopic structure of Pol III, consistent with transient binding to the pore (29Fernández-Tornero C. Böttcher B. Rashid U.J. Steuerwald U. Flörchinger B. Devos D.P. Lindner D. Müller C.W. EMBO J. 2010; 29: 3762-3772Crossref PubMed Scopus (56) Google Scholar). Third, C11 and A12.2 are required for transcription termination by Pol I and Pol III (10Chédin S. Riva M. Schultz P. Sentenac A. Carles C. Genes Dev. 1998; 12: 3857-3871Crossref PubMed Scopus (151) Google Scholar, 30Prescott E.M. Osheim Y.N. Jones H.S. Alen C.M. Roan J.G. Reeder R.H. Beyer A.L. Proudfoot N.J. Proc. Natl. Acad. Sci. U.S.A. 2004; 101: 6068-6073Crossref PubMed Scopus (59) Google Scholar), and the termination mechanism likely resembles that of archaeal polymerase (31Spitalny P. Thomm M. Mol. Microbiol. 2008; 67: 958-970Crossref PubMed Scopus (32) Google Scholar) but is different in Pol II. Fourth, the A12.2 and C11 C-ribbon domains may be able to swing between surface and pore locations because some density for the A12.2 C-ribbon was observed near the lobe in a Pol I EM reconstruction (3Kuhn C.D. Geiger S.R. Baumli S. Gartmann M. Gerber J. Jennebach S. Mielke T. Tschochner H. Beckmann R. Cramer P. Cell. 2007; 131: 1260-1272Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar) and because the strong Pol III cleavage can be even further enhanced by a mutation of the largest subunit that is predicted to disrupt a salt bridge between the Pol III counterpart of Rpb1 residue Asp-781 in the funnel domain F-loop and the C11 residue Arg-88 (corresponding to Rpb9 Arg-91) (8Thuillier V. Brun I. Sentenac A. Werner M. EMBO J. 1996; 15: 618-629Crossref PubMed Scopus (37) Google Scholar). It remains to be confirmed that A12.2 uses the same mechanism as C11. Unfortunately, replacing the Rpb9 C-ribbon with the A12.2 C-ribbon did not confer strong cleavage to Pol II (not shown), likely because Pol I has diverged much more from Pol II than Pol III.These results unveil the exceptional nature of Pol II, in contrast to Pol I, Pol III, and the archaeal polymerase, with respect to RNA cleavage. In the Pol II system, implementation of allosteric and direct cleavage stimulatory modes on two different proteins may have enabled new mechanisms of transcription regulation such as regulation by release of promoter-proximally stalled Pol II (18Cheung A.C. Cramer P. Nature. 2011; 471: 249-253Crossref PubMed Scopus (239) Google Scholar, 32Adelman K. Marr M.T. Werner J. Saunders A. Ni Z. Andrulis E.D. Lis J.T. Mol. Cell. 2005; 17: 103-112Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar, 33Palangat M. Renner D.B. Price D.H. Landick R. Proc. Natl. Acad. Sci. U.S.A. 2005; 102: 15036-15041Crossref PubMed Scopus (49) Google Scholar, 34Nechaev S. Fargo D.C. dos Santos G. Liu L. Gao Y. Adelman K. Science. 2010; 327: 335-338Crossref PubMed Scopus (304) Google Scholar). It may also have prevented premature Pol II termination at sites that would terminate Pol III and may have enabled elaborate 3′-end processing of Pol II transcripts. The weak intrinsic cleavage activity of Pol II may, however, suffice for proofreading after ubiquitous misincorporation events. IntroductionThe eukaryotic RNA polymerases Pol 3The abbreviations used are: Pol, RNA polymerase; TFS, transcription factor S; TFIIS, transcription factor IIS. I, II, and III share a highly conserved active center (1Cramer P. Bushnell D.A. Kornberg R.D. Science. 2001; 292: 1863-1876Crossref PubMed Scopus (955) Google Scholar, 2Jasiak A.J. Armache K.J. Martens B. Jansen R.P. Cramer P. Mol. Cell. 2006; 23: 71-81Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar, 3Kuhn C.D. Geiger S.R. Baumli S. Gartmann M. Gerber J. Jennebach S. Mielke T. Tschochner H. Beckmann R. Cramer P. Cell. 2007; 131: 1260-1272Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar) that catalyzes DNA-dependent RNA synthesis during gene transcription. The same active center also catalyzes cleavage of the nascent transcript during proofreading and recovery from transcription arrest (4Sosunov V. Sosunova E. Mustaev A. Bass I. Nikiforov V. Goldfarb A. EMBO J. 2003; 22: 2234-2244Crossref PubMed Scopus (161) Google Scholar, 5Kettenberger H. Armache K.J. Cramer P. Cell. 2003; 114: 347-357Abstract Full Text Full Text PDF PubMed Scopus (264) Google Scholar). Transcript cleavage is essential for cell viability (6Sigurdsson S. Dirac-Svejstrup A.B. Svejstrup J.Q. Mol. Cell. 2010; 38: 202-210Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). Despite the active center conservation, the strength of this intrinsic cleavage activity greatly differs between polymerases. Although the cleavage activity is very strong for Pol I (3Kuhn C.D. Geiger S.R. Baumli S. Gartmann M. Gerber J. Jennebach S. Mielke T. Tschochner H. Beckmann R. Cramer P. Cell. 2007; 131: 1260-1272Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar) and Pol III (7Alic N. Ayoub N. Landrieux E. Favry E. Baudouin-Cornu P. Riva M. Carles C. Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 10400-10405Crossref PubMed Scopus (42) Google Scholar, 8Thuillier V. Brun I. Sentenac A. Werner M. EMBO J. 1996; 15: 618-629Crossref PubMed Scopus (37) Google Scholar), it is very weak for Pol II. The molecular basis for this phenomenon remains unknown.Intrinsic transcript cleavage requires the homologous subunits A12.2, Rpb9, and C11 in Pol I, II, and III, respectively (3Kuhn C.D. Geiger S.R. Baumli S. Gartmann M. Gerber J. Jennebach S. Mielke T. Tschochner H. Beckmann R. Cramer P. Cell. 2007; 131: 1260-1272Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar, 9Walmacq C. Kireeva M.L. Irvin J. Nedialkov Y. Lubkowska L. Malagon F. Strathern J.N. Kashlev M. J. Biol. Chem. 2009; 284: 19601-19612Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, 10Chédin S. Riva M. Schultz P. Sentenac A. Carles C. Genes Dev. 1998; 12: 3857-3871Crossref PubMed Scopus (151) Google Scholar). Archaea contain a related protein, TFS, which is required for RNA cleavage by the polymerase (11Hausner W. Lange U. Musfeldt M. J. Biol. Chem. 2000; 275: 12393-12399Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar, 12Lange U. Hausner W. Mol. Microbiol. 2004; 52: 1133-1143Crossref PubMed Scopus (48) Google Scholar). All these proteins consist of two zinc-binding β-ribbon domains. Rpb9 resides on the enzyme surface, where its N-terminal zinc ribbon (N-ribbon) forms part of the Rpb1/9 jaw, and its C-terminal zinc ribbon (C-ribbon) binds between the Rpb1 funnel domain and the Rpb2 domains lobe and external-1 (Fig. 1). The very weak cleavage activity of Pol II is greatly stimulated by TFIIS, which contains a Pol II-binding domain that is located at the Rpb1/9 jaw and a C-ribbon (5Kettenberger H. Armache K.J. Cramer P. Cell. 2003; 114: 347-357Abstract Full Text Full Text PDF PubMed Scopus (264) Google Scholar, 13Qian X. Jeon C. Yoon H. Agarwal K. Weiss M.A. Nature. 1993; 365: 277-279Crossref PubMed Scopus (111) Google Scholar). The TFIIS C-ribbon binds the pore beneath the Pol II active center and reaches the active site with a hairpin containing the invariant residues Arg-287, Asp-290, and Glu-291 that are required for function (5Kettenberger H. Armache K.J. Cramer P. Cell. 2003; 114: 347-357Abstract Full Text Full Text PDF PubMed Scopus (264) Google Scholar, 14Jeon C. Yoon H. Agarwal K. Proc. Natl. Acad. Sci. U.S.A. 1994; 91: 9106-9110Crossref PubMed Scopus (71) Google Scholar, 15Awrey D.E. Shimasaki N. Koth C. Weilbaecher R. Olmsted V. Kazanis S. Shan X. Arellano J. Arrowsmith C.H. Kane C.M. Edwards A.M. J. Biol. Chem. 1998; 273: 22595-22605Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar, 16Kettenberger H. Armache K.J. Cramer P. Mol. Cell. 2004; 16: 955-965Abstract Full Text Full Text PDF PubMed Scopus (344) Google Scholar, 17Wang D. Bushnell D.A. Huang X. Westover K.D. Levitt M. Kornberg R.D. Science. 2009; 324: 1203-1206Crossref PubMed Scopus (183) Google Scholar, 18Cheung A.C. Cramer P. Nature. 2011; 471: 249-253Crossref PubMed Scopus (239) Google Scholar). Although A12.2 and C11 contain these three hairpin residues, Rpb9 lacks the residue corresponding to Glu-291. It remains unclear how the ribbon domains are related evolutionarily and mechanistically and how this may result in different cleavage activities. Here we used a combination of mutagenesis, cleavage assays, and x-ray crystallography to unravel the molecular basis for differential intrinsic RNA cleavage activities of Pol II and Pol III and suggest how the C-ribbon domains are related evolutionarily and how different cleavage activities arose during evolution.