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A Dual Interface Determines the Recognition of RNA Polymerase II by RNA Capping Enzyme*

2010; Elsevier BV; Volume: 285; Issue: 44 Linguagem: Inglês

10.1074/jbc.m110.145110

ISSN

1083-351X

Autores

Man-Hee Suh, Peter Meyer, Meigang Gu, Ping Ye, Mincheng Zhang, Craig D. Kaplan, Christopher D. Lima, Jianhua Fu,

Tópico(s)

RNA modifications and cancer

Resumo

RNA capping enzyme (CE) is recruited specifically to RNA polymerase II (Pol II) transcription sites to facilitate cotranscriptional 5′-capping of pre-mRNA and other Pol II transcripts. The current model to explain this specific recruitment of CE to Pol II as opposed to Pol I and Pol III rests on the interaction between CE and the phosphorylated C-terminal domain (CTD) of Pol II largest subunit Rpb1 and more specifically between the CE nucleotidyltransferase domain and the phosphorylated CTD. Through biochemical and diffraction analyses, we demonstrate the existence of a distinctive stoichiometric complex between CE and the phosphorylated Pol II (Pol IIO). Analysis of the complex revealed an additional and unexpected polymerase-CE interface (PCI) located on the multihelical Foot domain of Rpb1. We name this interface PCI1 and the previously known nucleotidyltransferase/phosphorylated CTD interface PCI2. Although PCI1 and PCI2 individually contribute to only weak interactions with CE, a dramatically stabilized and stoichiometric complex is formed when PCI1 and PCI2 are combined in cis as they occur in an intact phosphorylated Pol II molecule. Disrupting either PCI1 or PCI2 by alanine substitution or deletion diminishes CE association with Pol II and causes severe growth defects in vivo. Evidence from manipulating PCI1 indicates that the Foot domain contributes to the specificity in CE interaction with Pol II as opposed to Pol I and Pol III. Our results indicate that the dual interface based on combining PCI1 and PCI2 is required for directing CE to Pol II elongation complexes. RNA capping enzyme (CE) is recruited specifically to RNA polymerase II (Pol II) transcription sites to facilitate cotranscriptional 5′-capping of pre-mRNA and other Pol II transcripts. The current model to explain this specific recruitment of CE to Pol II as opposed to Pol I and Pol III rests on the interaction between CE and the phosphorylated C-terminal domain (CTD) of Pol II largest subunit Rpb1 and more specifically between the CE nucleotidyltransferase domain and the phosphorylated CTD. Through biochemical and diffraction analyses, we demonstrate the existence of a distinctive stoichiometric complex between CE and the phosphorylated Pol II (Pol IIO). Analysis of the complex revealed an additional and unexpected polymerase-CE interface (PCI) located on the multihelical Foot domain of Rpb1. We name this interface PCI1 and the previously known nucleotidyltransferase/phosphorylated CTD interface PCI2. Although PCI1 and PCI2 individually contribute to only weak interactions with CE, a dramatically stabilized and stoichiometric complex is formed when PCI1 and PCI2 are combined in cis as they occur in an intact phosphorylated Pol II molecule. Disrupting either PCI1 or PCI2 by alanine substitution or deletion diminishes CE association with Pol II and causes severe growth defects in vivo. Evidence from manipulating PCI1 indicates that the Foot domain contributes to the specificity in CE interaction with Pol II as opposed to Pol I and Pol III. Our results indicate that the dual interface based on combining PCI1 and PCI2 is required for directing CE to Pol II elongation complexes. IntroductionIn eukaryotic cells, RNA polymerase II (RNA Pol II) 5The abbreviations used are: RNA PolRNA polymeraseCEcapping enzymeCTDC-terminal repeated domainCTD-Pphosphorylated CTDNTnucleotidyltransferaseMBPmaltose-binding proteinPol IIOphosphorylated Pol IISECsize-exclusion chromatographyOBoligonucleotide bindingPCIpolymerase CE interfaceFCCFoot, nucleic acid cleft, and CTDFOA5-fluoroorotic acidTAPtandem affinity purification. and its associated factors carry out transcription of pre-mRNAs, snRNAs, telomerase RNA, and other noncoding RNAs. Pol II also couples transcription to nuclear processes including pre-mRNA modifications (1McCracken S. Fong N. Rosonina E. Yankulov K. Brothers G. Siderovski D. Hessel A. Foster S. Shuman S. Bentley D.L. Genes Dev. 1997; 11: 3306-3318Crossref PubMed Scopus (428) Google Scholar, 2McCracken S. Fong N. Yankulov K. Ballantyne S. Pan G. Greenblatt J. Patterson S.D. Wickens M. Bentley D.L.. Nature. 1997; 385: 357-361Crossref PubMed Scopus (735) Google Scholar, 3Cho E.J. Takagi T. Moore C.R. Buratowski S. Genes Dev. 1997; 11: 3319-3326Crossref PubMed Scopus (369) Google Scholar, 4Bentley D. Curr. Opin. Cell Biol. 1999; 11: 347-351Crossref PubMed Scopus (196) Google Scholar), mRNA export, and chromatin reconfiguration (5Buratowski S. Moazed D. Nature. 2005; 435: 1174-1175Crossref PubMed Scopus (10) Google Scholar, 6Phatnani H.P. Greenleaf A.L. Genes Dev. 2006; 20: 2922-2936Crossref PubMed Scopus (545) Google Scholar, 7Akhtar A. Gasser S.M. Nat. Rev. Genet. 2007; 8: 507-517Crossref PubMed Scopus (342) Google Scholar). Coupling RNA processing with synthesis is presumed to be critical in restricting the temporal window during which unmodified transcripts are vulnerable to degradation by endogenous ribonuclease activities (8Furuichi Y. LaFiandra A. Shatkin A.J. Nature. 1977; 266: 235-239Crossref PubMed Scopus (350) Google Scholar, 9Schwer B. Mao X. Shuman S. Nucleic Acids Res. 1998; 26: 2050-2057Crossref PubMed Scopus (84) Google Scholar, 10Jimeno-González S. Haaning L.L. Malagon F. Jensen T.H. Mol. Cell. 2010; 37: 580-587Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar) and directing RNA processing factors to sites of Pol II transcription at specific steps during Pol II progression through a gene. The Pol II elongation complex coordinates these transactions to help orchestrate control over gene expression (for review, see Ref. 11Perales R. Bentley D. Mol. Cell. 2009; 36: 178-191Abstract Full Text Full Text PDF PubMed Scopus (290) Google Scholar). Such coordination is mediated by specific and reversible interactions among Pol II and the factors involved in elongation.The formation of RNA 5′ cap structure, m7GpppN, is the first transcription-coordinated RNA modification event, and it occurs as soon as the transcript attains ∼25 nucleotides (12Coppola J.A. Field A.S. Luse D.S. Proc. Natl. Acad. Sci. U.S.A. 1983; 80: 1251-1255Crossref PubMed Scopus (71) Google Scholar, 13Hagler J. Shuman S. Science. 1992; 255: 983-986Crossref PubMed Scopus (67) Google Scholar, 14Rasmussen E.B. Lis J.T. Proc. Natl. Acad. Sci. U.S.A. 1993; 90: 7923-7927Crossref PubMed Scopus (280) Google Scholar, 15Mandal S.S. Chu C. Wada T. Handa H. Shatkin A.J. Reinberg D. Proc. Natl. Acad. Sci. U.S.A. 2004; 101: 7572-7577Crossref PubMed Scopus (133) Google Scholar). The RNA cap is formed in three enzymatic steps (16Shatkin A.J. Cell. 1976; 9: 645-653Abstract Full Text PDF PubMed Scopus (719) Google Scholar, 17Shuman S. Prog. Nucleic Acid Res. Mol. Biol. 2001; 66: 1-40Crossref PubMed Google Scholar); (i) removal of the 5′ γ-phosphate catalyzed by the RNA triphosphatase; (ii) attachment of a GMP to the 5′ diphosphate by the guanylyltransferase; (iii) methylation of the 5′ guanine by the cap methyltransferase. The first two of these steps are closely linked and coupled to Pol II transcription in most organisms. Mammals combine their triphosphatase and guanylyltransferase into a bi-functional capping enzyme (CE) encoded by a single gene. Fungal CEs comprise the two enzymes in a tightly associated complex, as exemplified by the Saccharomyces cerevisiae CE, a heterotetrameric complex consisting of the guanylyltransferase Ceg1 and triphosphatase Cet1 subunits (18Gu M. Rajashankar K.R. Lima C.D. Structure. 2010; 18: 216-227Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar) (for review, see Ref. 19Ghosh A. Lima C.D. Wiley Interdiscipl. Rev.: RNA. 2010; 1: 152-172Crossref PubMed Scopus (43) Google Scholar). Emerging evidence has also suggested non-catalytic roles of the CE in transcription regulation. For instance, stimulation of CE by HIV Tat promotes read-through of the viral genome (20Chiu Y.L. Ho C.K. Saha N. Schwer B. Shuman S. Rana T.M. Mol. Cell. 2002; 10: 585-597Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar, 21Zhou M. Deng L. Kashanchi F. Brady J.N. Shatkin A.J. Kumar A. Proc. Natl. Acad. Sci. U.S.A. 2003; 100: 12666-12671Crossref PubMed Scopus (60) Google Scholar). CE has been implicated in the formation of an early elongation checkpoint, as has been observed in 5′-paused Pol II complexes (15Mandal S.S. Chu C. Wada T. Handa H. Shatkin A.J. Reinberg D. Proc. Natl. Acad. Sci. U.S.A. 2004; 101: 7572-7577Crossref PubMed Scopus (133) Google Scholar, 22Kim H.J. Jeong S.H. Heo J.H. Jeong S.J. Kim S.T. Youn H.D. Han J.W. Lee H.W. Cho E.J. Mol. Cell. Biol. 2004; 24: 6184-6193Crossref PubMed Scopus (32) Google Scholar, 23Pei Y. Schwer B. Shuman S. J. Biol. Chem. 2003; 278: 7180-7188Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, 24Guiguen A. Soutourina J. Dewez M. Tafforeau L. Dieu M. Raes M. Vandenhaute J. Werner M. Hermand D. EMBO J. 2007; 26: 1552-1559Crossref PubMed Scopus (54) Google Scholar, 25Glover-Cutter K. Kim S. Espinosa J. Bentley D.L. Nat. Struct. Mol. Biol. 2008; 15: 71-78Crossref PubMed Scopus (248) Google Scholar). Cotranscriptional capping is effectuated by the specific interaction between a CE and its cognate RNA polymerase that supports a stoichiometric CE-polymerase complex (13Hagler J. Shuman S. Science. 1992; 255: 983-986Crossref PubMed Scopus (67) Google Scholar, 26Guarino L.A. Jin J. Dong W. J. Virol. 1998; 72: 10003-10010Crossref PubMed Google Scholar), as exemplified in certain viral transcription/processing systems in each of which the transcription of viral genes is mediated by a single virus-encoded RNA polymerase (27Broyles S.S. J. Gen. Virol. 2003; 84: 2293-2303Crossref PubMed Scopus (161) Google Scholar).The issue of class specificity in CE recruitment arises in eukaryotes because of the existence of three related RNA polymerases, Pol I, Pol II, and Pol III, that share five subunits (Rpb5, -6, -8, -10, and -12) but synthesize different classes of RNAs (28Woychik N.A. Liao S.M. Kolodziej P.A. Young R.A. Genes Dev. 1990; 4: 313-323Crossref PubMed Scopus (136) Google Scholar). Capping is specifically targeted to Pol II transcription complexes (1McCracken S. Fong N. Rosonina E. Yankulov K. Brothers G. Siderovski D. Hessel A. Foster S. Shuman S. Bentley D.L. Genes Dev. 1997; 11: 3306-3318Crossref PubMed Scopus (428) Google Scholar, 3Cho E.J. Takagi T. Moore C.R. Buratowski S. Genes Dev. 1997; 11: 3319-3326Crossref PubMed Scopus (369) Google Scholar) after the phosphorylation of Pol II by TFIIH during transcription initiation (29Rodriguez C.R. Cho E.J. Keogh M.C. Moore C.L. Greenleaf A.L. Buratowski S. Mol. Cell. Biol. 2000; 20: 104-112Crossref PubMed Scopus (161) Google Scholar). Importantly, mRNAs that are forced to be transcribed by Pol I, Pol III, or T7 RNA polymerase are uncapped (30Lo H.J. Huang H.K. Donahue T.F. Mol. Cell. Biol. 1998; 18: 665-675Crossref PubMed Scopus (39) Google Scholar, 31Gunnery S. Mathews M.B. Mol. Cell. Biol. 1995; 15: 3597-3607Crossref PubMed Scopus (53) Google Scholar, 32Dower K. Rosbash M. RNA. 2002; 8: 686-697Crossref PubMed Scopus (79) Google Scholar). The mechanism for the specific recruitment has been ascribed to the interaction between CE and the phosphorylated C-terminal repeated domain (CTD) of Rpb1, a domain that is unique to the Pol II largest subunit (1McCracken S. Fong N. Rosonina E. Yankulov K. Brothers G. Siderovski D. Hessel A. Foster S. Shuman S. Bentley D.L. Genes Dev. 1997; 11: 3306-3318Crossref PubMed Scopus (428) Google Scholar, 3Cho E.J. Takagi T. Moore C.R. Buratowski S. Genes Dev. 1997; 11: 3319-3326Crossref PubMed Scopus (369) Google Scholar, 33Yue Z. Maldonado E. Pillutla R. Cho H. Reinberg D. Shatkin A.J. Proc. Natl. Acad. Sci. U.S.A. 1997; 94: 12898-12903Crossref PubMed Scopus (196) Google Scholar, 34Ho C.K. Sriskanda V. McCracken S. Bentley D. Schwer B. Shuman S. J. Biol. Chem. 1998; 273: 9577-9585Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar). The absence of CTD in Pol I and Pol III (35Allison L.A. Moyle M. Shales M. Ingles C.J. Cell. 1985; 42: 599-610Abstract Full Text PDF PubMed Scopus (442) Google Scholar, 36Corden J.L. Cadena D.L. Ahearn Jr., J.M. Dahmus M.E. Proc. Natl. Acad. Sci. U.S.A. 1985; 82: 7934-7938Crossref PubMed Scopus (237) Google Scholar) suggests that the CTD imparts the Pol II apparatus with a unique ability to regulate interactions with various factors including the CE in a phosphorylation-dependent manner. It is known that CTD phosphorylation occurs reversibly on the serine-2, -5, or -7 residues in its heptapeptide motif, 1YSPTSPS7. It is also known that CTD phosphorylation patterns correlate with discrete stages of transcription (37Buratowski S. Nat. Struct. Biol. 2003; 10: 679-680Crossref PubMed Scopus (258) Google Scholar, 38Buratowski S. Mol. Cell. 2009; 36: 541-546Abstract Full Text Full Text PDF PubMed Scopus (540) Google Scholar). CE binding to Pol II is stimulated by the phosphorylation at either Ser-2 or Ser-5 positions in the CTD (39Ho C.K. Shuman S. Mol. Cell. 1999; 3: 405-411Abstract Full Text Full Text PDF PubMed Scopus (264) Google Scholar, 40Ursic D. Finkel J.S. Culbertson M.R. RNA Biol. 2008; 5: 1-4Crossref PubMed Scopus (6) Google Scholar), but Ser(P)-5 catalyzed by the Kin28 kinase of TFIIH predominates during early stages of transcription (41Komarnitsky P. Cho E.J. Buratowski S. Genes Dev. 2000; 14: 2452-2460Crossref PubMed Scopus (795) Google Scholar, 42Boehm A.K. Saunders A. Werner J. Lis J.T. Mol. Cell. Biol. 2003; 23: 7628-7637Crossref PubMed Scopus (187) Google Scholar, 43Kim M. Suh H. Cho E.J. Buratowski S. J. Biol. Chem. 2009; 284: 26421-26426Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar). The binding per se does not require the presence of transcript in Pol II, as has been observed either in the absence of RNA polymerization events (3Cho E.J. Takagi T. Moore C.R. Buratowski S. Genes Dev. 1997; 11: 3319-3326Crossref PubMed Scopus (369) Google Scholar) or with the purified polymerase (33Yue Z. Maldonado E. Pillutla R. Cho H. Reinberg D. Shatkin A.J. Proc. Natl. Acad. Sci. U.S.A. 1997; 94: 12898-12903Crossref PubMed Scopus (196) Google Scholar). Interestingly, the elongation factor Spt5 of the fission yeast Schizosaccharomyces pombe seems to contribute a CE recruitment function in vivo that overlaps with the function provided by the CTD (45Pei Y. Shuman S. J. Biol. Chem. 2002; 277: 19639-19648Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 46Schneider S. Pei Y. Shuman S. Schwer B. Mol. Cell. Biol. 2010; 30: 2353-2364Crossref PubMed Scopus (46) Google Scholar). Although evidence exists for a functional interaction between Spt5 and CE in the budding yeast S. cerevisiae (47Lindstrom D.L. Squazzo S.L. Muster N. Burckin T.A. Wachter K.C. Emigh C.A. McCleery J.A. Yates 3rd, J.R. Hartzog G.A. Mol. Cell. Biol. 2003; 23: 1368-1378Crossref PubMed Scopus (204) Google Scholar), it remains unclear if this interaction is critical in targeting CE to Pol II transcription because of a lack of biochemical and structural data on this issue. Because S. cerevisiae Spt5 also participates in Pol I transcription (48Schneider D.A. French S.L. Osheim Y.N. Bailey A.O. Vu L. Dodd J. Yates J.R. Beyer A.L. Nomura M. Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 12707-12712Crossref PubMed Scopus (76) Google Scholar), Spt5 is not likely to be a decisive factor in the specific recruitment of CE to Pol II in budding yeast, although it may contribute a salubrious effect on the proper assembly of CE into the early elongation complex.The current model for Pol II-linked cotranscriptional RNA 5′-capping rests on the binding of CE to the phosphorylated CTD (CTD-P) (1McCracken S. Fong N. Rosonina E. Yankulov K. Brothers G. Siderovski D. Hessel A. Foster S. Shuman S. Bentley D.L. Genes Dev. 1997; 11: 3306-3318Crossref PubMed Scopus (428) Google Scholar, 3Cho E.J. Takagi T. Moore C.R. Buratowski S. Genes Dev. 1997; 11: 3319-3326Crossref PubMed Scopus (369) Google Scholar, 33Yue Z. Maldonado E. Pillutla R. Cho H. Reinberg D. Shatkin A.J. Proc. Natl. Acad. Sci. U.S.A. 1997; 94: 12898-12903Crossref PubMed Scopus (196) Google Scholar, 34Ho C.K. Sriskanda V. McCracken S. Bentley D. Schwer B. Shuman S. J. Biol. Chem. 1998; 273: 9577-9585Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar). The CTD is present but disordered in the Pol II crystal structure and may extend from the globular core of Pol II into bulk solution (49Spåhr H. Calero G. Bushnell D.A. Kornberg R.D. Proc. Natl. Acad. Sci. U.S.A. 2009; 106: 9185-9190Crossref PubMed Scopus (38) Google Scholar, 50Cramer P. Bushnell D.A. Kornberg R.D. Science. 2001; 292: 1863-1876Crossref PubMed Scopus (955) Google Scholar). Structural studies of a complex between the Cgt1 subunit (Candida albicans homolog of S. cerevisiae Ceg1) and synthetic CTD heptads bearing Ser(P)-5 have revealed interactions with about 2.5 CTD repeats that are coordinated in a surface groove within the nucleotidyltransferase (NT) domain of Cgt1 (51Fabrega C. Shen V. Shuman S. Lima C.D. Mol. Cell. 2003; 11: 1549-1561Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar). Given that there are 26–27 heptad repeats in the CTD of S. cerevisiae and the CE structures are highly conserved between C. albicans and S. cerevisiae, one could expect at least eight copies of CE to bind, possibly distributively, along the CTD of yeast Pol II upon CTD phosphorylation. However, it is difficult to conceive that such spatially undefined interactions can provide the precise spatial and temporal information required to target CE activity to a narrow window within the transcription cycle. Furthermore, recent experiments with human cells found that the CTD alone was not sufficient for enhancing cotranscriptional processing in vivo, suggesting the involvement of additional Pol II components and/or associated factors in the process (52Natalizio B.J. Robson-Dixon N.D. Garcia-Blanco M.A. J. Biol. Chem. 2009; 284: 8692-8702Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). It, therefore, appears that CTD-P alone does not fully account for the Pol II specificity in CE recruitment even though CTD phosphorylation is critical for RNA capping in vivo.Here, we present evidence for a mechanism that complements the CTD-P-only recruitment model for CE function, thus shedding light on the Pol II-specificity in cotranscriptional targeting of the budding yeast CE. We demonstrate a distinctive and stoichiometric CE-Pol IIO complex that requires two interfaces on the polymerase. One interface is based on the CTD-P as expected from the common model and the Cgt1/CTD-P cocrystal structure (51Fabrega C. Shen V. Shuman S. Lima C.D. Mol. Cell. 2003; 11: 1549-1561Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar). The other interface deemed important for the recognition is formed with the Pol II multihelical Foot domain (50Cramer P. Bushnell D.A. Kornberg R.D. Science. 2001; 292: 1863-1876Crossref PubMed Scopus (955) Google Scholar). Mutational impairment of either interface disrupts the stability of the complex in vitro and compromises cell growth, whereas overexpression of CE suppresses the in vivo effects of the interface mutations. Mutational evidence also indicates that the Foot domain plays an essential role in imparting specificity toward Pol II as opposed to Pol I and Pol III. Our data indicate that the simultaneous formation of the dual interface is necessary and sufficient to stabilize CE binding to the polymerase. Hence, the dual interface is the physical basis for specifying RNA CE to Pol II transcription in vivo. We also show that each of the two interfaces alone is unable to support stable interactions with CE. Therefore, the post-initiation Pol II capping complex may dissociate as soon as the state of either interface is changed, a modus operandi that seems suitable for the dynamic assembly and disassembly of multiprotein complexes involved in transcription regulation.EXPERIMENTAL PROCEDURESRNA polymerases were purified directly from the budding yeast S. cerevisiae by applying the TAP-tagging strategy (53Puig O. Caspary F. Rigaut G. Rutz B. Bouveret E. Bragado-Nilsson E. Wilm M. Séraphin B. Methods. 2001; 24: 218-229Crossref PubMed Scopus (1415) Google Scholar) with the procedures given in the supplemental Experimental Procedures. Pol IIO was generated using the in vitro phosphorylation by MAP kinase 2 (MAPK2) under a condition known to produce both Ser(P)-2 and Ser(P)-5 sites but with preference toward Ser-5 positions (supplemental Experimental Procedures). The Pol IIO was indistinguishable from that generated with the yeast TFIIK kinase, a subcomplex of TFIIH (supplemental Fig. S1B), which is responsible for in vivo phosphorylation of the CTD and the recruitment of CE (29Rodriguez C.R. Cho E.J. Keogh M.C. Moore C.L. Greenleaf A.L. Buratowski S. Mol. Cell. Biol. 2000; 20: 104-112Crossref PubMed Scopus (161) Google Scholar, 54Hong S.W. Hong S.M. Yoo J.W. Lee Y.C. Kim S. Lis J.T. Lee D.K. Proc. Natl. Acad. Sci. U.S.A. 2009; 106: 14276-14280Crossref PubMed Scopus (31) Google Scholar). As such, the Pol IIO generated in vitro was suitable for binding studies with CE. The heterodimeric yeast CE and its individual subunits (Ceg1 and Cet1) were expressed in Escherichia coli and purified as described in the supplemental Experimental Procedures.Glutathione S-transferase (GST) and maltose-binding protein (MBP) fusions were constructed using PCR primers corresponding to sequence regions that each encompassed a Pol II structural domain (50Cramer P. Bushnell D.A. Kornberg R.D. Science. 2001; 292: 1863-1876Crossref PubMed Scopus (955) Google Scholar), i.e. the CTD of Rpb1, Rpb1-Foot, Rpb2-Lobe (residues 216–407), Rpb5, Rpb6, and Rpb8 subunits and Rpb6 truncations. GST-CTD-P and MBP-CTD-P were generated by the same in vitro phosphorylation procedure as used for generating Pol IIO. The GST- and MBP-mediated chromatographic procedures are described in the supplemental Experimental Procedures.Affinity capture by calmodulin-coupled beads was used in analyzing CE recognition of RNA polymerases. The calmodulin-binding peptide on the C terminus of a polymerase subunit (Rpb7 for Pol II, Rpb8 for Pol I) was derived from the TAP purification procedure. The calmodulin-binding peptide allowed the immobilization of a polymerase complex on calmodulin beads. Detailed conditions are given in the supplemental Experimental Procedures. Size exclusion chromatography was applied to assess stabilities of the various polymerase-CE complexes under the conditions given in the supplemental Experimental Procedures.For the determination of protein stoichiometry, the gels resulting from affinity assays and size-exclusion chromatography experiments were stained with Coomassie Blue and digitized with an optical scanner (CanoScan-4400F) that proved to produce an even background. Intensities of bands (e.g. Ceg1 and Rpb2 for CE and Pol II, respectively) were quantified with ImageQuant software (GE Healthcare), normalized against their respective molecular weights, and used to determine the stoichiometry.Western blotting was performed using antibodies directed against Ceg1 (55Schwer B. Shuman S. RNA. 1996; 2: 574-583PubMed Google Scholar) and Cet1 (56Myers L.C. Lacomis L. Erdjument-Bromage H. Tempst P. Mol. Cell. 2002; 10: 883-894Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar) for the yeast CE and against Rpb4 (NeoClone) for yeast Pol II. The bands were quantified using the ChemiDoc system (Bio-Rad).Plasmid shuffle assays were based on the counter-selection of URA3-containing cells by 5-fluoroorotic acid (FOA) and performed with standard procedures. Details of the shuffling experiment and also the Ceg1 overexpression in the strains are described in the supplemental Experimental Procedures. The coimmunoprecipitation experiments were done with cell extracts of FOA-resistant strains, each carrying an rpb1 allele, using the procedure given in the supplemental Experimental Procedures. X-ray crystallography was employed to reveal structural aspects of the interaction between CE and Pol II. Detailed procedures and conditions are given in the supplemental Experimental Procedures. IntroductionIn eukaryotic cells, RNA polymerase II (RNA Pol II) 5The abbreviations used are: RNA PolRNA polymeraseCEcapping enzymeCTDC-terminal repeated domainCTD-Pphosphorylated CTDNTnucleotidyltransferaseMBPmaltose-binding proteinPol IIOphosphorylated Pol IISECsize-exclusion chromatographyOBoligonucleotide bindingPCIpolymerase CE interfaceFCCFoot, nucleic acid cleft, and CTDFOA5-fluoroorotic acidTAPtandem affinity purification. and its associated factors carry out transcription of pre-mRNAs, snRNAs, telomerase RNA, and other noncoding RNAs. Pol II also couples transcription to nuclear processes including pre-mRNA modifications (1McCracken S. Fong N. Rosonina E. Yankulov K. Brothers G. Siderovski D. Hessel A. Foster S. Shuman S. Bentley D.L. Genes Dev. 1997; 11: 3306-3318Crossref PubMed Scopus (428) Google Scholar, 2McCracken S. Fong N. Yankulov K. Ballantyne S. Pan G. Greenblatt J. Patterson S.D. Wickens M. Bentley D.L.. Nature. 1997; 385: 357-361Crossref PubMed Scopus (735) Google Scholar, 3Cho E.J. Takagi T. Moore C.R. Buratowski S. Genes Dev. 1997; 11: 3319-3326Crossref PubMed Scopus (369) Google Scholar, 4Bentley D. Curr. Opin. Cell Biol. 1999; 11: 347-351Crossref PubMed Scopus (196) Google Scholar), mRNA export, and chromatin reconfiguration (5Buratowski S. Moazed D. Nature. 2005; 435: 1174-1175Crossref PubMed Scopus (10) Google Scholar, 6Phatnani H.P. Greenleaf A.L. Genes Dev. 2006; 20: 2922-2936Crossref PubMed Scopus (545) Google Scholar, 7Akhtar A. Gasser S.M. Nat. Rev. Genet. 2007; 8: 507-517Crossref PubMed Scopus (342) Google Scholar). Coupling RNA processing with synthesis is presumed to be critical in restricting the temporal window during which unmodified transcripts are vulnerable to degradation by endogenous ribonuclease activities (8Furuichi Y. LaFiandra A. Shatkin A.J. Nature. 1977; 266: 235-239Crossref PubMed Scopus (350) Google Scholar, 9Schwer B. Mao X. Shuman S. Nucleic Acids Res. 1998; 26: 2050-2057Crossref PubMed Scopus (84) Google Scholar, 10Jimeno-González S. Haaning L.L. Malagon F. Jensen T.H. Mol. Cell. 2010; 37: 580-587Abstract Full Text Full Text PDF PubMed Scopus (53) Google Scholar) and directing RNA processing factors to sites of Pol II transcription at specific steps during Pol II progression through a gene. The Pol II elongation complex coordinates these transactions to help orchestrate control over gene expression (for review, see Ref. 11Perales R. Bentley D. Mol. Cell. 2009; 36: 178-191Abstract Full Text Full Text PDF PubMed Scopus (290) Google Scholar). Such coordination is mediated by specific and reversible interactions among Pol II and the factors involved in elongation.The formation of RNA 5′ cap structure, m7GpppN, is the first transcription-coordinated RNA modification event, and it occurs as soon as the transcript attains ∼25 nucleotides (12Coppola J.A. Field A.S. Luse D.S. Proc. Natl. Acad. Sci. U.S.A. 1983; 80: 1251-1255Crossref PubMed Scopus (71) Google Scholar, 13Hagler J. Shuman S. Science. 1992; 255: 983-986Crossref PubMed Scopus (67) Google Scholar, 14Rasmussen E.B. Lis J.T. Proc. Natl. Acad. Sci. U.S.A. 1993; 90: 7923-7927Crossref PubMed Scopus (280) Google Scholar, 15Mandal S.S. Chu C. Wada T. Handa H. Shatkin A.J. Reinberg D. Proc. Natl. Acad. Sci. U.S.A. 2004; 101: 7572-7577Crossref PubMed Scopus (133) Google Scholar). The RNA cap is formed in three enzymatic steps (16Shatkin A.J. Cell. 1976; 9: 645-653Abstract Full Text PDF PubMed Scopus (719) Google Scholar, 17Shuman S. Prog. Nucleic Acid Res. Mol. Biol. 2001; 66: 1-40Crossref PubMed Google Scholar); (i) removal of the 5′ γ-phosphate catalyzed by the RNA triphosphatase; (ii) attachment of a GMP to the 5′ diphosphate by the guanylyltransferase; (iii) methylation of the 5′ guanine by the cap methyltransferase. The first two of these steps are closely linked and coupled to Pol II transcription in most organisms. Mammals combine their triphosphatase and guanylyltransferase into a bi-functional capping enzyme (CE) encoded by a single gene. Fungal CEs comprise the two enzymes in a tightly associated complex, as exemplified by the Saccharomyces cerevisiae CE, a heterotetrameric complex consisting of the guanylyltransferase Ceg1 and triphosphatase Cet1 subunits (18Gu M. Rajashankar K.R. Lima C.D. Structure. 2010; 18: 216-227Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar) (for review, see Ref. 19Ghosh A. Lima C.D. Wiley Interdiscipl. Rev.: RNA. 2010; 1: 152-172Crossref PubMed Scopus (43) Google Scholar). Emerging evidence has also suggested non-catalytic roles of the CE in transcription regulation. For instance, stimulation of CE by HIV Tat promotes read-through of the viral genome (20Chiu Y.L. Ho C.K. Saha N. Schwer B. Shuman S. Rana T.M. Mol. Cell. 2002; 10: 585-597Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar, 21Zhou M. Deng L. Kashanchi F. Brady J.N. Shatkin A.J. Kumar A. Proc. Natl. Acad. Sci. U.S.A. 2003; 100: 12666-12671Crossref PubMed Scopus (60) Google Scholar). CE has been implicated in the formation of an early elongation checkpoint, as has been observed in 5′-paused Pol II complexes (15Mandal S.S. Chu C. Wada T. Handa H. Shatkin A.J. Reinberg D. Proc. Natl. Acad. Sci. U.S.A. 2004; 101: 7572-7577Crossref PubMed Scopus (133) Google Scholar, 22Kim H.J. Jeong S.H. Heo J.H. Jeong S.J. Kim S.T. Youn H.D. Han J.W. Lee H.W. Cho E.J. Mol. Cell. Biol. 2004; 24: 6184-6193Crossref PubMed Scopus (32) Google Scholar, 23Pei Y. Schwer B. Shuman S. J. Biol. Chem. 2003; 278: 7180-7188Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, 24Guiguen A. Soutourina J. Dewez M. Tafforeau L. Dieu M. Raes M. Vandenhaute J. Werner M. Hermand D. EMBO J. 2007; 26: 1552-1559Crossref PubMed Scopus (54) Google Scholar, 25Glover-Cutter K. Kim S. Espinosa J. Bentley D.L. Nat. Struct. Mol. Biol. 2008; 15: 71-78Crossref PubMed Scopus (248) Google Scholar). Cotranscriptional capping is effectuated by the specific interaction between a CE and its cognate RNA polymerase that supports a stoichiometric CE-polymerase complex (13Hagler J. Shuman S. Science. 1992; 255: 983-986Crossref PubMed Scopus (67) Google Scholar, 26Guarino L.A. Jin J. Dong W. J. Virol. 1998; 72: 10003-10010Crossref PubMed Google Scholar), as exemplified in certain viral transcription/processing systems in each of which the transcription of viral genes is mediated by a single virus-encoded RNA polymerase (27Broyles S.S. J. Gen. Virol. 2003; 84: 2293-2303Crossref PubMed Scopus (161) Google Scholar).The issue of class specificity in CE recruitment arises in eukaryotes because of the existence of three related RNA polymerases, Pol I, Pol II, and Pol III, that share five subunits (Rpb5, -6, -8, -10, and -12) but synthesize different classes of RNAs (28Woychik N.A. Liao S.M. Kolodziej P.A. Young R.A. Genes Dev. 1990; 4: 313-323Crossref PubMed Scopus (136) Google Scholar). Capping is specifically targeted to Pol II transcription complexes (1McCracken S. Fong N. Rosonina E. Yankulov K. Brothers G. Siderovski D. Hessel A. Foster S. Shuman S. Bentley D.L. Genes Dev. 1997; 11: 3306-3318Crossref PubMed Scopus (428) Google Scholar, 3Cho E.J. Takagi T. Moore C.R. Buratowski S. Genes Dev. 1997; 11: 3319-3326Crossref PubMed Scopus (369) Google Scholar) after the phosphorylation of Pol II by TFIIH during transcription initiation (29Rodriguez C.R. Cho E.J. Keogh M.C. Moore C.L. Greenleaf A.L. Buratowski S. Mol. Cell. Biol. 2000; 20: 104-112Crossref PubMed Scopus (161) Google Scholar). Importantly, mRNAs that are forced to be transcribed by Pol I, Pol III, or T7 RNA polymerase are uncapped (30Lo H.J. Huang H.K. Donahue T.F. Mol. Cell. Biol. 1998; 18: 665-675Crossref PubMed Scopus (39) Google Scholar, 31Gunnery S. Mathews M.B. Mol. Cell. Biol. 1995; 15: 3597-3607Crossref PubMed Scopus (53) Google Scholar, 32Dower K. Rosbash M. RNA. 2002; 8: 686-697Crossref PubMed Scopus (79) Google Scholar). The mechanism for the specific recruitment has been ascribed to the interaction between CE and the phosphorylated C-terminal repeated domain (CTD) of Rpb1, a domain that is unique to the Pol II largest subunit (1McCracken S. Fong N. Rosonina E. Yankulov K. Brothers G. Siderovski D. Hessel A. Foster S. Shuman S. Bentley D.L. Genes Dev. 1997; 11: 3306-3318Crossref PubMed Scopus (428) Google Scholar, 3Cho E.J. Takagi T. Moore C.R. Buratowski S. Genes Dev. 1997; 11: 3319-3326Crossref PubMed Scopus (369) Google Scholar, 33Yue Z. Maldonado E. Pillutla R. Cho H. Reinberg D. Shatkin A.J. Proc. Natl. Acad. Sci. U.S.A. 1997; 94: 12898-12903Crossref PubMed Scopus (196) Google Scholar, 34Ho C.K. Sriskanda V. McCracken S. Bentley D. Schwer B. Shuman S. J. Biol. Chem. 1998; 273: 9577-9585Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar). The absence of CTD in Pol I and Pol III (35Allison L.A. Moyle M. Shales M. Ingles C.J. Cell. 1985; 42: 599-610Abstract Full Text PDF PubMed Scopus (442) Google Scholar, 36Corden J.L. Cadena D.L. Ahearn Jr., J.M. Dahmus M.E. Proc. Natl. Acad. Sci. U.S.A. 1985; 82: 7934-7938Crossref PubMed Scopus (237) Google Scholar) suggests that the CTD imparts the Pol II apparatus with a unique ability to regulate interactions with various factors including the CE in a phosphorylation-dependent manner. It is known that CTD phosphorylation occurs reversibly on the serine-2, -5, or -7 residues in its heptapeptide motif, 1YSPTSPS7. It is also known that CTD phosphorylation patterns correlate with discrete stages of transcription (37Buratowski S. Nat. Struct. Biol. 2003; 10: 679-680Crossref PubMed Scopus (258) Google Scholar, 38Buratowski S. Mol. Cell. 2009; 36: 541-546Abstract Full Text Full Text PDF PubMed Scopus (540) Google Scholar). CE binding to Pol II is stimulated by the phosphorylation at either Ser-2 or Ser-5 positions in the CTD (39Ho C.K. Shuman S. Mol. Cell. 1999; 3: 405-411Abstract Full Text Full Text PDF PubMed Scopus (264) Google Scholar, 40Ursic D. Finkel J.S. Culbertson M.R. RNA Biol. 2008; 5: 1-4Crossref PubMed Scopus (6) Google Scholar), but Ser(P)-5 catalyzed by the Kin28 kinase of TFIIH predominates during early stages of transcription (41Komarnitsky P. Cho E.J. Buratowski S. Genes Dev. 2000; 14: 2452-2460Crossref PubMed Scopus (795) Google Scholar, 42Boehm A.K. Saunders A. Werner J. Lis J.T. Mol. Cell. Biol. 2003; 23: 7628-7637Crossref PubMed Scopus (187) Google Scholar, 43Kim M. Suh H. Cho E.J. Buratowski S. J. Biol. Chem. 2009; 284: 26421-26426Abstract Full Text Full Text PDF PubMed Scopus (104) Google Scholar). The binding per se does not require the presence of transcript in Pol II, as has been observed either in the absence of RNA polymerization events (3Cho E.J. Takagi T. Moore C.R. Buratowski S. Genes Dev. 1997; 11: 3319-3326Crossref PubMed Scopus (369) Google Scholar) or with the purified polymerase (33Yue Z. Maldonado E. Pillutla R. Cho H. Reinberg D. Shatkin A.J. Proc. Natl. Acad. Sci. U.S.A. 1997; 94: 12898-12903Crossref PubMed Scopus (196) Google Scholar). Interestingly, the elongation factor Spt5 of the fission yeast Schizosaccharomyces pombe seems to contribute a CE recruitment function in vivo that overlaps with the function provided by the CTD (45Pei Y. Shuman S. J. Biol. Chem. 2002; 277: 19639-19648Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 46Schneider S. Pei Y. Shuman S. Schwer B. Mol. Cell. Biol. 2010; 30: 2353-2364Crossref PubMed Scopus (46) Google Scholar). Although evidence exists for a functional interaction between Spt5 and CE in the budding yeast S. cerevisiae (47Lindstrom D.L. Squazzo S.L. Muster N. Burckin T.A. Wachter K.C. Emigh C.A. McCleery J.A. Yates 3rd, J.R. Hartzog G.A. Mol. Cell. Biol. 2003; 23: 1368-1378Crossref PubMed Scopus (204) Google Scholar), it remains unclear if this interaction is critical in targeting CE to Pol II transcription because of a lack of biochemical and structural data on this issue. Because S. cerevisiae Spt5 also participates in Pol I transcription (48Schneider D.A. French S.L. Osheim Y.N. Bailey A.O. Vu L. Dodd J. Yates J.R. Beyer A.L. Nomura M. Proc. Natl. Acad. Sci. U.S.A. 2006; 103: 12707-12712Crossref PubMed Scopus (76) Google Scholar), Spt5 is not likely to be a decisive factor in the specific recruitment of CE to Pol II in budding yeast, although it may contribute a salubrious effect on the proper assembly of CE into the early elongation complex.The current model for Pol II-linked cotranscriptional RNA 5′-capping rests on the binding of CE to the phosphorylated CTD (CTD-P) (1McCracken S. Fong N. Rosonina E. Yankulov K. Brothers G. Siderovski D. Hessel A. Foster S. Shuman S. Bentley D.L. Genes Dev. 1997; 11: 3306-3318Crossref PubMed Scopus (428) Google Scholar, 3Cho E.J. Takagi T. Moore C.R. Buratowski S. Genes Dev. 1997; 11: 3319-3326Crossref PubMed Scopus (369) Google Scholar, 33Yue Z. Maldonado E. Pillutla R. Cho H. Reinberg D. Shatkin A.J. Proc. Natl. Acad. Sci. U.S.A. 1997; 94: 12898-12903Crossref PubMed Scopus (196) Google Scholar, 34Ho C.K. Sriskanda V. McCracken S. Bentley D. Schwer B. Shuman S. J. Biol. Chem. 1998; 273: 9577-9585Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar). The CTD is present but disordered in the Pol II crystal structure and may extend from the globular core of Pol II into bulk solution (49Spåhr H. Calero G. Bushnell D.A. Kornberg R.D. Proc. Natl. Acad. Sci. U.S.A. 2009; 106: 9185-9190Crossref PubMed Scopus (38) Google Scholar, 50Cramer P. Bushnell D.A. Kornberg R.D. Science. 2001; 292: 1863-1876Crossref PubMed Scopus (955) Google Scholar). Structural studies of a complex between the Cgt1 subunit (Candida albicans homolog of S. cerevisiae Ceg1) and synthetic CTD heptads bearing Ser(P)-5 have revealed interactions with about 2.5 CTD repeats that are coordinated in a surface groove within the nucleotidyltransferase (NT) domain of Cgt1 (51Fabrega C. Shen V. Shuman S. Lima C.D. Mol. Cell. 2003; 11: 1549-1561Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar). Given that there are 26–27 heptad repeats in the CTD of S. cerevisiae and the CE structures are highly conserved between C. albicans and S. cerevisiae, one could expect at least eight copies of CE to bind, possibly distributively, along the CTD of yeast Pol II upon CTD phosphorylation. However, it is difficult to conceive that such spatially undefined interactions can provide the precise spatial and temporal information required to target CE activity to a narrow window within the transcription cycle. Furthermore, recent experiments with human cells found that the CTD alone was not sufficient for enhancing cotranscriptional processing in vivo, suggesting the involvement of additional Pol II components and/or associated factors in the process (52Natalizio B.J. Robson-Dixon N.D. Garcia-Blanco M.A. J. Biol. Chem. 2009; 284: 8692-8702Abstract Full Text Full Text PDF PubMed Scopus (24) Google Scholar). It, therefore, appears that CTD-P alone does not fully account for the Pol II specificity in CE recruitment even though CTD phosphorylation is critical for RNA capping in vivo.Here, we present evidence for a mechanism that complements the CTD-P-only recruitment model for CE function, thus shedding light on the Pol II-specificity in cotranscriptional targeting of the budding yeast CE. We demonstrate a distinctive and stoichiometric CE-Pol IIO complex that requires two interfaces on the polymerase. One interface is based on the CTD-P as expected from the common model and the Cgt1/CTD-P cocrystal structure (51Fabrega C. Shen V. Shuman S. Lima C.D. Mol. Cell. 2003; 11: 1549-1561Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar). The other interface deemed important for the recognition is formed with the Pol II multihelical Foot domain (50Cramer P. Bushnell D.A. Kornberg R.D. Science. 2001; 292: 1863-1876Crossref PubMed Scopus (955) Google Scholar). Mutational impairment of either interface disrupts the stability of the complex in vitro and compromises cell growth, whereas overexpression of CE suppresses the in vivo effects of the interface mutations. Mutational evidence also indicates that the Foot domain plays an essential role in imparting specificity toward Pol II as opposed to Pol I and Pol III. Our data indicate that the simultaneous formation of the dual interface is necessary and sufficient to stabilize CE binding to the polymerase. Hence, the dual interface is the physical basis for specifying RNA CE to Pol II transcription in vivo. We also show that each of the two interfaces alone is unable to support stable interactions with CE. Therefore, the post-initiation Pol II capping complex may dissociate as soon as the state of either interface is changed, a modus operandi that seems suitable for the dynamic assembly and disassembly of multiprotein complexes involved in transcription regulation.

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