RNA Polymerase II Holoenzymes and Subcomplexes

TATA-binding protein general transcription factor cyclin-dependent kinase polymerase C-terminal domain preinitiation complex. The RNA polymerase II holoenzyme is the form of eukaryotic RNA polymerase II that is recruited to the promoters of protein-coding genes in living cells. The exact composition of the holoenzyme is not entirely established, due in part to technical difficulties associated with purifying intact megadalton size multiprotein complexes. Nonetheless, yeast and human holoenzyme preparations have been described that consist of near stoichiometric levels of most components known to be generally involved in initiation other than TATA-binding protein (TBP)1 and its associated factors. We review here the functions of five major components of yeast RNA polymerase II holoenzymes: core RNA polymerase II, the general transcription factors (GTFs), the core Srb-mediator complex, the Srb10 cyclin-dependent kinase (CDK) complex, and the Swi-Snf complex (Table I).Table IRNA polymerase II holoenzyme components in S. cerevisiaeFactorGeneSubunitEssential?FeaturesRefs.kDaRNA polymerase IIRPB1192YHeptapeptide repeat71Young R.A. Davis R.W. Science. 1983; 222: 778-782Crossref PubMed Scopus (602) Google Scholar, 72Ingles C.J. Himmelfarb H.J. Shales M. Greenleaf A.L. Friesen J.D. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 2157-2161Crossref PubMed Scopus (20) Google Scholar, 73Allison L.A. Moyle M. Shales M. Ingles C.J. Cell. 1985; 42: 599-610Abstract Full Text PDF PubMed Scopus (443) Google ScholarRPB2139Y74Sweetser D. Nonet M. Young R.A. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 1192-1196Crossref PubMed Scopus (256) Google ScholarRPB335Y75Kolodziej P. Young R.A. Mol. Cell. Biol. 1989; 9: 5387-5394Crossref PubMed Scopus (67) Google ScholarRPB425N76Woychik N.A. Young R.A. Mol. Cell. Biol. 1989; 9: 2854-2859Crossref PubMed Scopus (149) Google ScholarRPB525YShared with PolI, II, III77Woychik N.A. Liao S.M. Kolodziej P.A. Young R.A. Genes Dev. 1990; 4: 313-323Crossref PubMed Scopus (138) Google ScholarRPB618YShared with PolI, II, III77Woychik N.A. Liao S.M. Kolodziej P.A. Young R.A. Genes Dev. 1990; 4: 313-323Crossref PubMed Scopus (138) Google Scholar, 78Woychik N.A. Young R.A. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 3999-4003Crossref PubMed Scopus (21) Google ScholarRPB719Y79McKune K. Richards K.L. Edwards A.M. Young R.A. Woychik N.A. Yeast. 1993; 9: 295-299Crossref PubMed Scopus (76) Google ScholarRPB817YShared with PolI, II, III77Woychik N.A. Liao S.M. Kolodziej P.A. Young R.A. Genes Dev. 1990; 4: 313-323Crossref PubMed Scopus (138) Google ScholarRPB914N80Woychik N.A. Lane W.S. Young R.A. J. Biol. Chem. 1991; 266: 19053-19055Abstract Full Text PDF PubMed Google ScholarRPB108YShared with PolI, II, III81Woychik N.A. Young R.A. J. Biol. Chem. 1990; 265: 17816-17819Abstract Full Text PDF PubMed Google ScholarRPB1114Y82Woychik N.A. McKune K. Lane W.S. Young R.A. Gene Expr. 1993; 3: 77-82PubMed Google ScholarRPB128YShared with PolI, II, III83Treich I. Carles C. Riva M. Sentenac A. Gene Expr. 1992; 2: 31-37PubMed Google ScholarTFIIHTFB173YNucleotide excision repair84Gileadi O. Feaver W.J. Kornberg R.D. Science. 1992; 257: 1389-1392Crossref PubMed Scopus (53) Google ScholarTFB259YNucleotide excision repair85Feaver W.J. Henry N.L. Wang Z. Wu X. Svejstrup J.Q. Bushnell D.A. Friedberg E.C. Kornberg R.D. J. Biol. Chem. 1997; 272: 19319-19327Abstract Full Text Full Text PDF PubMed Scopus (62) Google ScholarTFB332YNucleotide excision repair85Feaver W.J. Henry N.L. Wang Z. Wu X. Svejstrup J.Q. Bushnell D.A. Friedberg E.C. Kornberg R.D. J. Biol. Chem. 1997; 272: 19319-19327Abstract Full Text Full Text PDF PubMed Scopus (62) Google ScholarTFB437Y85Feaver W.J. Henry N.L. Wang Z. Wu X. Svejstrup J.Q. Bushnell D.A. Friedberg E.C. Kornberg R.D. J. Biol. Chem. 1997; 272: 19319-19327Abstract Full Text Full Text PDF PubMed Scopus (62) Google ScholarRAD390YDNA helicase86Feaver W.J. Svejstrup J.Q. Bardwell L. Bardwell A.J. Buratowski S. Gulyas K.D. Donahue T.F. Friedberg E.C. Kornberg R.D. Cell. 1993; 75: 1379-1387Abstract Full Text PDF PubMed Scopus (283) Google ScholarSSL152YNucleotide excision repair86Feaver W.J. Svejstrup J.Q. Bardwell L. Bardwell A.J. Buratowski S. Gulyas K.D. Donahue T.F. Friedberg E.C. Kornberg R.D. Cell. 1993; 75: 1379-1387Abstract Full Text PDF PubMed Scopus (283) Google ScholarSSL2/RAD2595YDNA helicase87Svejstrup J.Q. Wang Z. Feaver W.J. Wu X. Bushnell D.A. Donahue T.F. Friedberg E.C. Kornberg R.D. Cell. 1995; 80: 21-28Abstract Full Text PDF PubMed Scopus (239) Google ScholarKIN2835YCyclin-dependent CTD kinase88Feaver W.J. Svejstrup J.Q. Henry N.L. Kornberg R.D. Cell. 1994; 79: 1103-1109Abstract Full Text PDF PubMed Scopus (359) Google ScholarCCL145YKin28 cyclin partner89Svejstrup J.Q. Feaver W.J. J. Biol. Chem. 1996; 271: 643-645Abstract Full Text Full Text PDF PubMed Scopus (31) Google ScholarTFIIETFA155Y90Feaver W.J. Henry N.L. Bushnell D.A. Sayre M.H. Brickner J.H. Gileadi O. Kornberg R.D. J. Biol. Chem. 1994; 269: 27549-27553Abstract Full Text PDF PubMed Google ScholarTFA237Y90Feaver W.J. Henry N.L. Bushnell D.A. Sayre M.H. Brickner J.H. Gileadi O. Kornberg R.D. J. Biol. Chem. 1994; 269: 27549-27553Abstract Full Text PDF PubMed Google ScholarTFIIFSSU1/TFG182Y91Henry N.L. Campbell A.M. Feaver W.J. Poon D. Weil P.A. Kornberg R.D. Genes Dev. 1994; 8: 2868-2878Crossref PubMed Scopus (125) Google ScholarTFG247Y91Henry N.L. Campbell A.M. Feaver W.J. Poon D. Weil P.A. 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Biol. 1996; 16: 3308-3316Crossref PubMed Scopus (137) Google Scholar Open table in a new tab The holoenzyme concept stems from the discovery that Srb proteins are critical for regulated transcription of protein coding genes and the observation that these proteins are tightly associated with a portion of core RNA polymerase II in yeast cells (1Koleske A.J. Young R.A. Nature. 1994; 368: 466-469Crossref PubMed Scopus (531) Google Scholar). The genes encoding the yeast Srb proteins were discovered through a genetic screen designed to identify components of the transcription apparatus that are involved in the response to transcriptional regulators (2Nonet M.L. Young R.A. Genetics. 1989; 123: 715-724Crossref PubMed Google Scholar, 3Koleske A.J. Young R.A. Trends Biochem. Sci. 1995; 20: 113-116Abstract Full Text PDF PubMed Scopus (266) Google Scholar). Attempts to purify these proteins led to the isolation of a large complex containing core RNA polymerase II, a subset of the general transcription factors, and a variety of regulatory proteins (1Koleske A.J. Young R.A. Nature. 1994; 368: 466-469Crossref PubMed Scopus (531) Google Scholar). This holoenzyme complex had the capacity to initiate transcription and respond to activators when supplemented with additional purified general transcription factorsin vitro. A subcomplex dissociated from the holoenzyme, which contains the Srb and additional proteins, reconstituted the response to activators in a defined in vitro transcription system (4Kim Y.-J. Bjorklund S. Li Y. Sayre M.H. Kornberg R.D. Cell. 1994; 77: 599-608Abstract Full Text PDF PubMed Scopus (885) Google Scholar). The response to activators is especially significant asin vitro systems reconstituted with yeast GTFs and polymerase alone are not activator-responsive (5Flanagan P.M. Kelleher III, R.J. Sayre M.H. Tschochner H. Kornberg R.D. Nature. 1991; 350: 436-438Crossref PubMed Scopus (260) Google Scholar, 6Flanagan P.M. Kelleher III, R.J. Tschochner H. Sayre M.H. Kornberg R.D. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 7659-7663Crossref PubMed Scopus (24) Google Scholar). Two of the yeast Srb proteins were found to be required for transcription of most protein-coding genes, and because they are found tightly associated with the holoenzyme, it seems likely that the Srb-containing holoenzyme is the form of RNA polymerase II that is recruited to most promotersin vivo (7Thompson C.M. Young R.A. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 4587-4590Crossref PubMed Scopus (209) Google Scholar). RNA polymerase II holoenzymes have been purified from many eukaryotic organisms (1Koleske A.J. Young R.A. Nature. 1994; 368: 466-469Crossref PubMed Scopus (531) Google Scholar, 4Kim Y.-J. Bjorklund S. Li Y. Sayre M.H. Kornberg R.D. 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Greenblatt J. J. Biol. Chem. 1997; 272: 24563-24571Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar), whereas other protocols generate holoenzymes in which only a single GTF (TFIIF) remains associated (4Kim Y.-J. Bjorklund S. Li Y. Sayre M.H. Kornberg R.D. Cell. 1994; 77: 599-608Abstract Full Text PDF PubMed Scopus (885) Google Scholar). Some yeast holoenzyme preparations contain stoichiometric levels of Swi-Snf (17Wilson C.J. Chao D.M. Imbalzano A.N. Schnitzler G.R. Kingston R.E. Young R.A. Cell. 1996; 84: 235-244Abstract Full Text Full Text PDF PubMed Scopus (324) Google Scholar), whereas others lack any detectable Swi-Snf protein (18Cairns B.R. Lorch Y. Li Y. Zhang M. Lacomis L. Erdjument-Bromage H. Tempst P. Du J. Laurent B. Kornberg R.D. Cell. 1996; 87: 1249-1260Abstract Full Text Full Text PDF PubMed Scopus (577) Google Scholar). In the present discussion, we will make the simplifying assumption that holoenzymes in living cells resemble the more complex preparations. Thus, the holoenzyme we discuss here is composed of core RNA polymerase II, all the GTFs other than TBP (and its associated proteins), the core Srb-mediator complex, the Srb10 cyclin-dependent kinase complex, and the Swi-Snf complex (Table I). Eukaryotic core RNA polymerase II (Pol II) was first purified by using transcription assays with promoterless templates (19Sawadogo M. Sentenac A. Annu. Rev. Biochem. 1990; 59: 711-754Crossref PubMed Scopus (309) Google Scholar, 20Young R.A. Annu. Rev. Biochem. 1991; 60: 689-715Crossref PubMed Scopus (367) Google Scholar). The purified enzyme typically has 10–12 subunits and is incapable of specific promoter recognition. Yeast RNA polymerase II consists of 12 subunits, RPB1–RPB12, which range in size from approximately 6 to 200 kDa. A very similar 12-subunit enzyme can be purified from human cells, and numerous subunit-subunit interactions within the polymerase have been delineated (21Acker J. de Graaff M. Cheynel I. Khazak V. Kedinger C. Vigneron M. J. Biol. Chem. 1997; 272: 16815-16821Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). An interesting feature of the enzyme is the highly conserved domain at the C terminus of the largest subunit (CTD). This domain contains multiple repeats of the consensus sequence YSPTSPS and, as discussed below, is a substrate for several kinases that have roles in regulation of gene expression. There is considerable evidence that the eukaryotic enzymes are highly conserved. Core Pol II molecules purified from a wide range of eukaryotes have conserved subunit structure and sequence. Moreover, many yeast Pol II subunit genes can be replaced with their mammalian counterparts in living cells without deleterious effects on cell function (22McKune K. Woychik N.A. 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Five such factors were found to be essential for this activity on most promoters used in vitro: TFIID, TFIIB, TFIIF, TFIIH, and TFIIE. As with core RNA polymerase, the GTFs are highly conserved among eukaryotes. The precise roles of the GTFs have been reviewed in detail elsewhere (27Orphanides G. Lagrange T. Reinberg D. Genes Dev. 1996; 10: 2657-2683Crossref PubMed Scopus (848) Google Scholar) and will not be discussed here. Based on the genetic and biochemical evidence to date, we favor the model that transcription activation at many promoters involves recruitment of the transcription initiation apparatus in two steps. In this model, recruitment of two complexes is required for activation: a TBP-containing complex (reviewed in Ref. 28Lee T.I. Young R.A. Genes Dev. 1998; 12: 1398-1408Crossref PubMed Scopus (156) Google Scholar) and a holoenzyme containing the remaining GTFs. Although recruitment of either complex could be sufficient to assemble the other, activators may target members from both complexes and/or multiple components within a single complex. This combination of multiple activator-target interactions would result in transcription. This model does not exclude the possibility that transcription initiation at some promoters involves recruitment of components in many steps, and initiation at other promoters can involve recruitment of the entire apparatus in a single step. Indeed, given the large number of promoters present in living cells and the diverse mechanisms known to regulate gene expression, it seems likely that the entire spectrum of possibilities is realized. Transcription reactions reconstituted in vitro with highly purified RNA polymerase II and GTFs are not responsive to activators. The yeast mediator is a multiprotein complex that was purified for its ability to mediate activation (4Kim Y.-J. Bjorklund S. Li Y. Sayre M.H. 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