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• W2039836615 abstract "Purification of multiprotein complexes such as transcription factor (TF) IIH and RNA polymerase II (pol II) has been a tedious task by conventional chromatography. To facilitate the purification, we have developed an effective scheme that allows human TFIIH and pol II to be isolated from HeLa-derived cell lines that conditionally express the FLAG-tagged p62 subunit of human TFIIH and the RPB9 subunit of human pol II, respectively. An approximate 2000-fold enrichment of FLAG-tagged TFIIH and a 1000-fold enhancement of total pol II are achieved by a one-step immunoaffinity purification. The purified complexes are functional in mediating basal and activated transcription, regardless of whether TATA-binding protein or TFIID is used as the TATA-binding factor. Interestingly, repression of basal transcription by the positive cofactor PC4 is alleviated by increasing amounts of TFIID, TFIIH, and pol II holoenzyme, suggesting that phosphorylation of PC4 by these proteins may cause a conformational change in the structure of PC4 that allows for preinitiation complex formation and initiation of transcription. Furthermore, pol II complexes with different phosphorylation states on the carboxyl-terminal domain of the largest subunit are selectively purified from the inducible pol II cell line, making it possible to dissect the role of carboxyl-terminal domain phosphorylation in the transcription process in a highly defined in vitrotranscription system. Purification of multiprotein complexes such as transcription factor (TF) IIH and RNA polymerase II (pol II) has been a tedious task by conventional chromatography. To facilitate the purification, we have developed an effective scheme that allows human TFIIH and pol II to be isolated from HeLa-derived cell lines that conditionally express the FLAG-tagged p62 subunit of human TFIIH and the RPB9 subunit of human pol II, respectively. An approximate 2000-fold enrichment of FLAG-tagged TFIIH and a 1000-fold enhancement of total pol II are achieved by a one-step immunoaffinity purification. The purified complexes are functional in mediating basal and activated transcription, regardless of whether TATA-binding protein or TFIID is used as the TATA-binding factor. Interestingly, repression of basal transcription by the positive cofactor PC4 is alleviated by increasing amounts of TFIID, TFIIH, and pol II holoenzyme, suggesting that phosphorylation of PC4 by these proteins may cause a conformational change in the structure of PC4 that allows for preinitiation complex formation and initiation of transcription. Furthermore, pol II complexes with different phosphorylation states on the carboxyl-terminal domain of the largest subunit are selectively purified from the inducible pol II cell line, making it possible to dissect the role of carboxyl-terminal domain phosphorylation in the transcription process in a highly defined in vitrotranscription system. RNA polymerase II general transcription factor transcription factor TATA-binding protein TBP-associated factor positive cofactor 4 preinitiation complex carboxyl-terminal domain column volume dithiothreitol phenylmethylsulfonyl fluoride. In vitro, RNA polymerase II (pol II)1 in association with general transcription factors (GTFs) TFIIB, TFIID, TFIIE, TFIIF, and TFIIH are capable of supporting basal transcription from most eukaryotic promoters (1Orphanides G. Lagrange T. Reinberg D. Genes Dev. 1996; 10: 2657-2683Crossref PubMed Scopus (849) Google Scholar, 2Roeder R.G. Trends Biochem. Sci. 1996; 21: 327-335Abstract Full Text PDF PubMed Scopus (718) Google Scholar, 3Wu S.-Y. Kershnar E. Chiang C.-M. EMBO J. 1998; 17: 4478-4490Crossref PubMed Scopus (44) Google Scholar). The previously defined GTF, TFIIA, has recently been shown to function as a coactivator in a highly purified cell-free transcription system devoid of the pol II-specific TBP-associated factors (TAFIIs) of TFIID, human mediator and other negative cofactors (3Wu S.-Y. Kershnar E. Chiang C.-M. EMBO J. 1998; 17: 4478-4490Crossref PubMed Scopus (44) Google Scholar). The GTFs and pol II can assemble on a class II promoter via the sequential assembly pathway (1Orphanides G. Lagrange T. Reinberg D. Genes Dev. 1996; 10: 2657-2683Crossref PubMed Scopus (849) Google Scholar, 2Roeder R.G. Trends Biochem. Sci. 1996; 21: 327-335Abstract Full Text PDF PubMed Scopus (718) Google Scholar) or via the recruitment of a preassembled pol II holoenzyme complex (4Koleske A.J. Young R.A. Nature. 1994; 368: 466-469Crossref PubMed Scopus (531) Google Scholar, 5Kim Y.-J. Björkland S. Li Y. Sayre M.H. Kornberg R.D. Cell. 1994; 77: 599-608Abstract Full Text PDF PubMed Scopus (886) Google Scholar, 6Ossipow V. Tassan J.-P. Nigg E.A. Schibler U. Cell. 1995; 83: 137-146Abstract Full Text PDF PubMed Scopus (178) Google Scholar, 7Maldonado E. Shiekhattar R. Sheldon M. Cho H. Drapkin R. Rickert P. Lees E. Anderson C.W. Linn S. Reinberg D. Nature. 1996; 381: 86-89Crossref PubMed Scopus (306) Google Scholar, 8Scully R. Anderson S.F. Chao D.M. Wei W. Ye L. Young R.A. Livingston D.M. Parvin J.D. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 5605-5610Crossref PubMed Scopus (422) Google Scholar, 9Pan G. Aso T. Greenblatt J. J. Biol. Chem. 1997; 272: 24563-24571Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 10Wu S.-Y. Chiang C.-M. J. Biol. Chem. 1998; 273: 12492-12498Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). In the sequential assembly pathway, TFIID, a multiprotein complex comprising TBP and TAFIIs, binds to the TATA box in the promoter region allowing for the recruitment of TFIIB, which then acts as a molecular bridge in recruiting the pol II·TFIIF complex. Next, TFIIE binds to pol II and in turn recruits TFIIH. Although the GTFs and pol II are able to support basal transcription in cell-free transcription systems, many other protein factors are also involved in vivo. These proteins work collectively in defining the efficiency of transcriptional initiation, elongation, and termination of eukaryotic genes transcribed by pol II. pol II is composed of 10–12 polypeptides ranging in size from 220 to 7 kDa, depending on the source of purification (11Young R. Annu. Rev. Biochem. 1991; 60: 689-715Crossref PubMed Scopus (368) Google Scholar, 12Acker 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, 13Khazak V. Estojak J. Cho H. Majors J. Sonoda G. Testa J.R. Golemis E.A. Mol. Cell. Biol. 1998; 18: 1935-1945Crossref PubMed Scopus (58) Google Scholar). The subunits of human pol II (or RNA polymerase B) have been defined as RPB1 (220 kDa), RPB2 (140 kDa), RPB3 (33 kDa), RPB4 (18 kDa), RPB5 (28 kDa), RPB6 (19 kDa), RPB7 (27 kDa), RPB8 (17 kDa), RPB9 (14.5 kDa), RPB10α (or RPB12, 7.0 kDa), RPB10β (or RPB10, 7.6 kDa), and RPB11 (14 kDa) (3Wu S.-Y. Kershnar E. Chiang C.-M. EMBO J. 1998; 17: 4478-4490Crossref PubMed Scopus (44) Google Scholar,11Young R. Annu. Rev. Biochem. 1991; 60: 689-715Crossref PubMed Scopus (368) Google Scholar, 12Acker 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, 13Khazak V. Estojak J. Cho H. Majors J. Sonoda G. Testa J.R. Golemis E.A. Mol. Cell. Biol. 1998; 18: 1935-1945Crossref PubMed Scopus (58) Google Scholar). RPB5, RPB6, RPB8, RPB10α, and RPB10β are shared by all three eukaryotic RNA polymerases, whereas the rest of the RPB components are unique to pol II (11Young R. Annu. Rev. Biochem. 1991; 60: 689-715Crossref PubMed Scopus (368) Google Scholar, 14Treich I. Carles C. Riva M. Sentenac A. Gene Exp. 1992; 2: 31-37PubMed Google Scholar). The two large subunits, RPB1 and RPB2, show sequence similarity with the β′ and β subunits of bacterial RNA polymerase, respectively (11Young R. Annu. Rev. Biochem. 1991; 60: 689-715Crossref PubMed Scopus (368) Google Scholar), and form an extended DNA-binding channel that surrounds the transcription start site (15Kim T.K. Lagrange T. Wang Y.H. Griffith J.D. Reinberg D. Ebright R.H. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 12268-12273Crossref PubMed Scopus (91) Google Scholar,16Fu J. Gerstein M. David P.R. Gnatt A.L. Bushnell D.A. Edwards A.M. Kornberg R.D. J. Mol. Biol. 1998; 280: 317-322Crossref PubMed Scopus (9) Google Scholar). In humans, RPB3 and RPB5 can each homodimerize and interact with almost all of the other subunits in vitro, suggesting that RPB3 and RPB5 may play a role in the assembly of pol II (12Acker 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). However, the isolation of an RPB3-deficient yeast pol II indicates that RPB3, which shares sequence similarity with the α subunit of prokaryotic RNA polymerase, is dispensable for complex assembly in vivo(17Svetlov V. Nolan K. Burgess R.R. J. Biol. Chem. 1998; 273: 10827-10830Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar). The finding that human RPB5 can functionally interact with the pX protein of the hepatitis B virus and with TFIIB also suggests that RPB5 may act as a bridging factor between pol II and regulatory proteins (18Cheong J. Yi M. Lin Y. Murakami S. EMBO J. 1995; 14: 143-150Crossref PubMed Scopus (241) Google Scholar, 19Haviv I. Shamay M. Doitsh G. Shaul Y. Mol. Cell. Biol. 1998; 18: 1562-1569Crossref PubMed Scopus (128) Google Scholar). In yeast, RPB4 and RPB7 form a dissociable subcomplex, which is preferentially incorporated into pol II during stationary phase induced by heat shock or nutrient stress (20Choder M. Young R.A. Mol. Cell. Biol. 1993; 13: 6984-6991Crossref PubMed Scopus (115) Google Scholar). Structural comparison and surface plasmon resonance measurements between wild type and RPB4/RPB7-deficient yeast pol II complexes suggest that RPB4 and RPB7 help stabilize the preinitiation complex on the promoter DNA under stress response in vivo (21Jensen G.J. Meredith G. Bushnell D.A. Kornberg R.D. EMBO J. 1998; 17: 2353-2358Crossref PubMed Scopus (58) Google Scholar). RPB9 is required for correct start site selection, as mutations in yeast RPB9 cause a shift in the transcription start site (22Hull M.W. McKune K. Woychik N.A. Genes Dev. 1995; 9: 481-490Crossref PubMed Scopus (87) Google Scholar). Interestingly, a suppressor mutation in RPB9 is able to compensate for the downstream shift in start site selection associated with TFIIB mutants, indicating that RPB9 can functionally interact with TFIIB in specifying the accurate transcription start site (23Sun Z.W. Tessmer A. Hampsey M. Nucleic Acids Res. 1996; 24: 2560-2566Crossref PubMed Scopus (50) Google Scholar). In addition, RPB9 seems to influence the efficiency of pol II elongation via its recognition of a pause signal on the DNA template (24Awrey D.E. Weilbaecher R.G. Hemming S.A. Orlicky S.M. Kane C.M. Edwards A.M. J. Biol. Chem. 1997; 272: 14747-14754Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar). Although the genes coding for the subunits of pol II from humans, Saccharomyces cerevisiae, andSchizosaccharomyces pombe have been cloned, sequenced, and all subunits excluding RPB4 and RPB9 have been shown to be essential in yeast (25Woychik N.A. Young R.A. Mol. Cell. Biol. 1989; 9: 2854-2859Crossref PubMed Scopus (149) Google Scholar, 26Woychik N.A. Lane W.S. Young R.A. J. Biol. Chem. 1991; 266: 19053-19055Abstract Full Text PDF PubMed Google Scholar), the function of many pol II subunits remains unanswered. Immunoprecipitation of 32P-labeled yeast extracts indicated that three of the pol II subunits, RPB1, RPB2, and RPB6, are phosphorylated in vivo (27Kolodziej P.A. Woychik N. Liao S.M. Young R.A. Mol. Cell. Biol. 1990; 10: 1915-1920Crossref PubMed Scopus (100) Google Scholar). The largest subunit of pol II, RPB1, contains consensus heptapeptide repeats of Tyr-Ser-Pro-Thr-Ser-Pro-Ser in its carboxyl-terminal domain (CTD) that occur 26 times in yeast and 52 times in humans (28Dahmus M.E. J. Biol. Chem. 1996; 271: 19009-19012Abstract Full Text Full Text PDF PubMed Scopus (462) Google Scholar). Phosphorylation of the CTD results in a hyperphosphorylated form of RPB1 (IIo), which is mostly found in actively elongating pol II complexes (28Dahmus M.E. J. Biol. Chem. 1996; 271: 19009-19012Abstract Full Text Full Text PDF PubMed Scopus (462) Google Scholar, 29Dahmus M.E. Prog. Nucleic Acids Res. Mol. Biol. 1994; 48: 143-179Crossref PubMed Scopus (87) Google Scholar, 30Svejstrup J.Q. Li Y. Fellows J. Gnatt A. Bjorklund S. Kornberg R.D. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 6075-6078Crossref PubMed Scopus (101) Google Scholar), and has been shown to associate with capping and processing factors (reviewed in Ref. 31Neugebauer K.M. Roth M. Genes Dev. 1997; 11: 3279-3285Crossref PubMed Scopus (102) Google Scholar). The pol II complex containing an unmodified or hypophosphorylated form of RPB1 (IIa) interacts with TBP, TFIIE, and mediator, and is preferentially assembled into a functional preinitiation complex (PIC) on the promoter (28Dahmus M.E. J. Biol. Chem. 1996; 271: 19009-19012Abstract Full Text Full Text PDF PubMed Scopus (462) Google Scholar, 29Dahmus M.E. Prog. Nucleic Acids Res. Mol. Biol. 1994; 48: 143-179Crossref PubMed Scopus (87) Google Scholar, 30Svejstrup J.Q. Li Y. Fellows J. Gnatt A. Bjorklund S. Kornberg R.D. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 6075-6078Crossref PubMed Scopus (101) Google Scholar, 32Lu H. Flores O. Weinmann R. Reinberg D. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 10004-10008Crossref PubMed Scopus (248) Google Scholar, 33Usheva A. Maldonado E. Goldring A. Lu H. Houbavi C. Reinberg D. Aloni Y. Cell. 1992; 69: 871-881Abstract Full Text PDF PubMed Scopus (179) Google Scholar, 34Maxon M.E. Goodrich J.A. Tjian R. Genes Dev. 1994; 8: 515-524Crossref PubMed Scopus (137) Google Scholar). In addition, a third form of pol II containing RPB1 without the CTD (IIb), presumably due to limited proteolysis, has also been identified (29Dahmus M.E. Prog. Nucleic Acids Res. Mol. Biol. 1994; 48: 143-179Crossref PubMed Scopus (87) Google Scholar). Phosphorylation of the CTD is a necessary step during the transition from the PIC to a stable elongation complex in many but not all promoters (35Serizawa H. Conaway J.W. Conaway R.C. Nature. 1993; 363: 371-374Crossref PubMed Scopus (139) Google Scholar, 36O'Brien T. Hardin S. Greenleaf A. Lis J.T. Nature. 1994; 370: 75-77Crossref PubMed Scopus (285) Google Scholar). Many protein kinases such as casein kinases I and II, cdc2 kinase, DNA-dependent protein kinase, c-Abl tyrosine kinase, mitogen-activated kinases ERK1 and ERK2, cdk8-cyclin C, P-TEFb, and TFIIH are able to phosphorylate the CTD on different phosphoacceptors (29Dahmus M.E. Prog. Nucleic Acids Res. Mol. Biol. 1994; 48: 143-179Crossref PubMed Scopus (87) Google Scholar, 37Lu H. Zawel L. Fisher L. Egly J.-M. Reinberg D. Nature. 1992; 358: 641-645Crossref PubMed Scopus (330) Google Scholar, 38Baskaran R. Dahmus M.E. Wang J.Y.J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 11167-11171Crossref PubMed Scopus (188) Google Scholar, 39Trigon S. Serizawa H. Conaway J.W. Conaway R.C. Jackson S.P. Morange M. J. Biol. Chem. 1998; 273: 6769-6775Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar, 40Hengartner C.J. Myer V.E. Liao S.-M. Wilson C.J. Koh S.S. Young R.A. Mol. Cell. 1998; 2: 43-53Abstract Full Text Full Text PDF PubMed Scopus (335) Google Scholar, 41Jones K.A. Genes Dev. 1997; 11: 2593-2599Crossref PubMed Scopus (196) Google Scholar). Although the exact phosphorylation sites of the CTD are not clear in vivo, the CTD has been shown to be highly phosphorylated mostly at the serines in the second and fifth position, as well as at the tyrosine and threonine residues (29Dahmus M.E. Prog. Nucleic Acids Res. Mol. Biol. 1994; 48: 143-179Crossref PubMed Scopus (87) Google Scholar, 38Baskaran R. Dahmus M.E. Wang J.Y.J. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 11167-11171Crossref PubMed Scopus (188) Google Scholar,42Zhang J. Corden J.L. J. Biol. Chem. 1991; 266: 2290-2296Abstract Full Text PDF PubMed Google Scholar, 43West M.L. Corden J.L. Genetics. 1995; 140: 1223-1233Crossref PubMed Google Scholar). It is likely that substrate specificity and temporal phosphorylation by various CTD kinases may functionally regulate pol II activity to selectively activate or suppress specific gene expression (40Hengartner C.J. Myer V.E. Liao S.-M. Wilson C.J. Koh S.S. Young R.A. Mol. Cell. 1998; 2: 43-53Abstract Full Text Full Text PDF PubMed Scopus (335) Google Scholar, 44Lee D.-K. Lis J.T. Nature. 1998; 393: 389-392Crossref PubMed Scopus (84) Google Scholar). One of the factors responsible for CTD phosphorylation is TFIIH, which preferentially phosphorylates a serine residue located in the fifth position of an oligomerized heptapeptide CTD sequence (39Trigon S. Serizawa H. Conaway J.W. Conaway R.C. Jackson S.P. Morange M. J. Biol. Chem. 1998; 273: 6769-6775Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar, 40Hengartner C.J. Myer V.E. Liao S.-M. Wilson C.J. Koh S.S. Young R.A. Mol. Cell. 1998; 2: 43-53Abstract Full Text Full Text PDF PubMed Scopus (335) Google Scholar). TFIIH is a large protein complex, composed of at least nine subunits (p89, p80, p62, p52, p44, p40 (cdk7), p36 (cyclin H), p34, and p32 (MAT1)), that has ATP-dependent DNA helicase, ATPase, protein kinase, and nucleotide-excision repair activities (reviewed in Ref.45Svejstrup J.Q. Vichi P. Egly J.-M. Trends Biochem. Sci. 1996; 21: 346-350Abstract Full Text PDF PubMed Scopus (196) Google Scholar). The two large subunits of TFIIH, p89 and p80, unwind the DNA in a 3′ → 5′ and 5′ → 3′ direction, respectively, making TFIIH a bidirectional DNA helicase (46Schaeffer L. Moncollin V. Roy R. Staub A. Mezzina M. Sarasin A. Weeda G. Hoeijmakers J.H.J. Egly J.-M. EMBO J. 1994; 13: 2388-2392Crossref PubMed Scopus (333) Google Scholar). In addition, mutations in the yeast homologs of human p89/ERCC3/XPB (SSL2), p80/ERCC2/XPD (RAD3), p62 (TFB1), p52 (TFB2), and p44 (SSL1) also cause a defect in nucleotide excision repair, indicating that TFIIH is involved in the coupling of transcription and DNA repair (reviewed in Refs. 45Svejstrup J.Q. Vichi P. Egly J.-M. Trends Biochem. Sci. 1996; 21: 346-350Abstract Full Text PDF PubMed Scopus (196) Google Scholar, 47Sancar A. Annu. Rev. Biochem. 1996; 65: 43-81Crossref PubMed Scopus (963) Google Scholar, and 48Feaver 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 Scholar). The kinase activity of TFIIH resides in the catalytic subunit cdk7, whose activity is modulated by cyclin H, MAT1, and other protein factors such as TFIIE and the mediator complex (reviewed in Ref. 45Svejstrup J.Q. Vichi P. Egly J.-M. Trends Biochem. Sci. 1996; 21: 346-350Abstract Full Text PDF PubMed Scopus (196) Google Scholar). cdk7 also forms a subcomplex, called CAK (cdk-activatingkinase), with cyclin H and MAT1. When associated with the other TFIIH components, CAK more efficiently phosphorylates the CTD, as compared with the free form of CAK (49Yankulov K.Y. Bentley D.L. EMBO J. 1997; 16: 1638-1646Crossref PubMed Scopus (153) Google Scholar). In addition to phosphorylating the CTD, cdk7 is required for mitosis and for the activation of many cdk/cyclin pairs involved in cell cycle progression (50Larochelle S. Pandur J. Fisher R.P. Salz H.K. Suter B. Genes Dev. 1998; 12: 370-381Crossref PubMed Scopus (148) Google Scholar, 51Harper J.W. Elledge S.J. Genes Dev. 1998; 12: 285-289Crossref PubMed Scopus (125) Google Scholar). The enzymatic activities of TFIIH are also involved in the transcription process. The ATP-dependent helicase activity of TFIIH is required for the opening of the promoter DNA surrounding the transcription start site, as well as the maintenance of the open complex (52Jiang Y. Yan M. Gralla J.D. J. Biol. Chem. 1995; 270: 27332-27338Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar, 53Holstege F.C.P. Fiedler U. Timmers H.T.M. EMBO J. 1997; 16: 7468-7480Crossref PubMed Scopus (158) Google Scholar). Formation of the first phosphodiester bond is not necessarily dependent on TFIIH, but is stimulated by its presence (54Goodrich J.A. Tjian R. Cell. 1994; 77: 145-156Abstract Full Text PDF PubMed Scopus (287) Google Scholar,55Kumar K.P. Akoulitchev S. Reinberg D. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 9767-9772Crossref PubMed Scopus (66) Google Scholar). However, TFIIH and ATP hydrolysis are clearly required for release of pol II from the promoter region (53Holstege F.C.P. Fiedler U. Timmers H.T.M. EMBO J. 1997; 16: 7468-7480Crossref PubMed Scopus (158) Google Scholar, 54Goodrich J.A. Tjian R. Cell. 1994; 77: 145-156Abstract Full Text PDF PubMed Scopus (287) Google Scholar, 55Kumar K.P. Akoulitchev S. Reinberg D. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 9767-9772Crossref PubMed Scopus (66) Google Scholar, 56Kugel J.F. Goodrich J.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 9232-9237Crossref PubMed Scopus (59) Google Scholar, 57Dvir A. Conaway R.C. Conaway J.W. J. Biol. Chem. 1996; 271: 23352-23356Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar, 58Dvir A. Conaway J.W. Conaway R.C. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 9006-9010Crossref PubMed Scopus (113) Google Scholar). In the absence of TFIIH, pol II tends to stall on the promoter-proximal region, leading to abortive transcription products (55Kumar K.P. Akoulitchev S. Reinberg D. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 9767-9772Crossref PubMed Scopus (66) Google Scholar, 58Dvir A. Conaway J.W. Conaway R.C. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 9006-9010Crossref PubMed Scopus (113) Google Scholar). The amount of the promoter-stalled pol II complex, nevertheless, is significantly reduced in the presence of TFIIH in an ATP-dependent manner (55Kumar K.P. Akoulitchev S. Reinberg D. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 9767-9772Crossref PubMed Scopus (66) Google Scholar,58Dvir A. Conaway J.W. Conaway R.C. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 9006-9010Crossref PubMed Scopus (113) Google Scholar). A stably elongating pol II complex often requires a hyperphosphorylated CTD that occurs subsequent to PIC assembly (28Dahmus M.E. J. Biol. Chem. 1996; 271: 19009-19012Abstract Full Text Full Text PDF PubMed Scopus (462) Google Scholar). The CTD kinase activity of TFIIH can be further stimulated by transcriptional activators that functionally interact with TFIIH (41Jones K.A. Genes Dev. 1997; 11: 2593-2599Crossref PubMed Scopus (196) Google Scholar). In addition to its central role in the transition from the initiation to the elongation phase, TFIIH can also function as a coactivator in supporting TBP-mediated activation, independent of TAFIIs (3Wu S.-Y. Kershnar E. Chiang C.-M. EMBO J. 1998; 17: 4478-4490Crossref PubMed Scopus (44) Google Scholar). To define the molecular mechanism of pol II and TFIIH in the transcription process and to overcome the difficulty in purifying these two multiprotein complexes by conventional chromatography, we have developed an effective method for the purification of human pol II and TFIIH. By using epitope-tagging and stable cell line approaches, we are able to purify nearly homogeneous preparations of human pol II and TFIIH by a one-step immunoaffinity purification. The purified multiprotein complexes contain all previously defined polypeptides and are devoid of other GTFs commonly copurified with pol II and TFIIH by traditional approaches. In conjunction with other protein factors and cofactors, the immunoaffinity-purified pol II and TFIIH are able to mediate both basal and activator-dependent transcription in a cell-free system reconstituted with either TBP or TFIID. Interestingly, in this highly purified in vitrotranscription system, repression of basal transcription by the positive cofactor PC4 can be alleviated by increasing amounts of TFIIH, TFIID, and a preassembled pol II holoenzyme complex but not by the other GTFs or pol II. We demonstrate that these multisubunit complexes can phosphorylate PC4, indicating that phosphorylation of PC4 by TFIID, TFIIH, or pol II holoenzyme may cause a conformational change in the structure of PC4 that allows for preinitiation complex formation and initiation of transcription. Furthermore, transcriptionally active pol II complexes enriched with either IIo or IIa forms of the CTD can be selectively isolated from the nuclear pellet of the inducible pol II cell line, providing a unique opportunity to dissect the role of CTD phosphorylation in the transcription process in a highly definedin vitro transcription system. A HeLa-derived cell line, F:62(H)-8, that conditionally expresses the FLAG-epitope tagged 62 kDa subunit of human TFIIH has been established (59Wu S.-Y. Chiang C.-M. BioTechniques. 1996; 21: 718-725Crossref PubMed Scopus (18) Google Scholar) and used for the purification of FLAG-tagged human TFIIH. Approximately 125 ml of F:62(H)-8 cells were expanded in Joklik media supplemented with 5% calf serum, and selected with tetracycline (1 μg/ml) and G418 (0.6 mg/ml total weight) for 3 days. After further expansion to 12 liters in tetracycline-containing medium without G418, F:62(H)-8 cells were pelleted at 1000 rpm, washed with 1× phosphate-buffered saline (four times), and resuspended in 9 liters of Joklik medium containing 5% calf serum. Four days after protein induction, nuclear extracts (∼60 ml) were prepared from F:62(H)-8 cells (∼60 liters) as described (60Dignam J.D. Lebovitz R.M. Roeder R.G. Nucleic Acids Res. 1983; 11: 1475-1489Crossref PubMed Scopus (9160) Google Scholar) and loaded onto a 60-ml phosphocellulose column (P11, Whatman) at a flow rate of 1 column volume (CV)/h. The column was washed with 3 CV of BC100 buffer (20% glycerol, 20 mm Tris-HCl, pH 7.9, at 4 °C, 0.2 mm EDTA, 0.5 mm PMSF, 1.0 mm DTT, and 100 mm KCl) and step eluted with 2.5 CV each of BC300, BC500, and BC850 (61Chiang C.-M. Ge H. Wang Z. Hoffman A. Roeder R.G. EMBO J. 1993; 12: 2749-2762Crossref PubMed Scopus (171) Google Scholar). The 0.5 m KCl fraction (termed P.5) containing the majority of FLAG-tagged TFIIH was dialyzed against 4 liters of BC100 for 5 h, and then centrifuged at 14,000 rpm for 15 min to remove insoluble materials. FLAG-tagged TFIIH in the P.5 supernatant was further concentrated by the addition of solid (NH4)2SO4 to 38% saturation (0.23 g/ml) and pelleted at 14,000 rpm for 15 min. The pellet was then resuspended in BC100 (2 ml of BC100 for every 10 ml of P.5 that was precipitated) and dialyzed against 4 liters of BC100 for 5 h to remove residual ammonium sulfate. Immunoaffinity purification of FLAG-tagged TFIIH was then performed by incubating approximately 300 μl of anti-FLAG M2 monoclonal antibody-conjugated agarose beads (Kodak/IBI) with 14 ml of nuclear extracts, 14 ml of the P.5 fraction, or 6 ml of the resuspended ammonium sulfate pellet at 4 °C for 6–12 h with constant rotation. The M2 agarose with bound proteins was sequentially washed five times each with BC300 plus 0.5 murea and 0.1% Nonidet P-40, BC100 (10 ml for each wash), and finally transferred to a 2-ml microcentrifuge spin column (Invitrogen). The bound proteins were incubated with 300 μl of BC100 plus 0.01% Nonidet P-40 containing 0.2 mg/ml FLAG peptide at 4 °C for 1 h with constant rotation (61Chiang C.-M. Ge H. Wang Z. Hoffman A. Roeder R.G. EMBO J. 1993; 12: 2749-2762Crossref PubMed Scopus (171) Google Scholar). Elutions were repeated for a total of three times. Purified proteins were then aliquoted and stored at −80 °C after snap freezing in liquid nitrogen. To purify FLAG-tagged human pol II, the hRPB9–3 cell line that conditionally expresses the FLAG-tagged RPB9 subunit of human pol II (10Wu S.-Y. Chiang C.-M. J. Biol. Chem. 1998; 273: 12492-12498Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar) was expanded to 60 liters with Joklik medium containing 5% calf serum as described above for the F:62(H)-8 cell line. Nuclear extract, cytoplasmic S100 fraction, and nuclear pellet were then prepared from the induced culture following the published protocols (60Dignam J.D. Lebovitz R.M. Roeder R.G. Nucleic Acids Res. 1983; 11: 1475-1489Crossref PubMed Scopus (9160) Google Scholar). To purify pol II from the soluble fractions, 60 ml of S100 (or nuclear extracts) was loaded onto a 60-ml phosphocellulose column and sequentially eluted with BC100, BC300, BC500, and BC850 as described above. Immunoaffinity purification of FLAG-tagged pol II was then conducted by incubating 14 ml of the S100 (or nuclear extracts) or P.5 fraction with 400 μl of M2-agarose as outlined above for FLAG-tagged TFIIH purification, except that BC850 plus 1.0 m urea and 0.1% Nonidet P-40 was used for the initial wash. To purify FLAG-tagged pol II from the chromosomal fraction, we adapted a previous procedure for initial pol II isolation (62Reinberg D. Roeder R.G. J. Biol. Chem. 1987; 262: 3310-3321Abstract Full Text PDF PubMed Google Scholar). Briefly, 35 ml of the hRPB9–3 nuclear pellet was slowly agitated with 70 ml of buffer B (50 mm Tris-HCl, pH 7.9 at 4 °C, 25% glycerol, 5 mm MgCl2, 5 mm EDTA, 5 mm EGTA, 5 mm DTT, 0.5 mm PMSF) and then with 11 ml of 3 m(NH4)2SO4 to adjust the ammonium sulfate concentration to 0.3 m. The mixture was stirred for additional 30 min and then sonicated for 5 × 1 min with 20-s intervals. After spinning at 40,000 rpm for 90 min, the supernatant was adjusted to 0.1 m ammonium sulfate concentration by gradually adding 2 volumes of buffer B with syringe. The precipitated material was removed by centrifugation at 40,000 rpm for 60 min. pol II was then precipitated by adding solid ammonium sulfate to 65% saturation (i.e. 0.42 g/ml of suspension) a" @default.
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• W2039836615 date "1998-12-01" @default.
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• W2039836615 title "Immunoaffinity Purification and Functional Characterization of Human Transcription Factor IIH and RNA Polymerase II from Clonal Cell Lines That Conditionally Express Epitope-tagged Subunits of the Multiprotein Complexes" @default.
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• W2039836615 doi "https://doi.org/10.1074/jbc.273.51.34444" @default.
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