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• W2082616562 abstract "The multisubunit Mediator is a well established transcription coactivator for gene-specific activators. However, recent studies have shown that, although not essential for basal transcription by purified RNA polymerase II (pol II) and general initiation factors, Mediator is essential for basal transcription in nuclear extracts that contain a more physiological complement of factors (Mittler, G., Kremmer, E., Timmers, H. T., and Meisterernst, M. (2001) EMBO Rep. 2, 808–813; Baek, H. J., Malik, S., Qin, J., and Roeder, R. G. (2002) Mol. Cell. Biol. 22, 2842–2852). Here, mechanistic studies with immobilized DNA templates, purified factors, and factor-depleted HeLa extracts have shown (i) that Mediator enhancement of basal transcription correlates with Mediator-dependent recruitment of pol II and general initiation factors (transcription factor (TF) IIB and TFIIE) to the promoter; (ii) that Mediator and TFIIB, which both interact with pol II, are jointly required for pol II recruitment to the promoter and that TFIIB recruitment is Mediator-dependent, whereas Mediator recruitment is TFIIB-independent; (iii) that a high level of TFIIB can bypass the Mediator requirement for basal transcription and pol II recruitment in nuclear extract, thus indicating a conditional restriction of TFIIB function and a key role of Mediator in overcoming this restriction; and (iv) that an earlier rate-limiting step involves formation of a TFIID-Mediator-promoter complex. These results support a stepwise assembly model, rather than a preformed holoenzyme model, for Mediator-dependent assembly of a basal preinitiation complex and, more important, identify a step involving TFIIB as a key site of action of Mediator. The multisubunit Mediator is a well established transcription coactivator for gene-specific activators. However, recent studies have shown that, although not essential for basal transcription by purified RNA polymerase II (pol II) and general initiation factors, Mediator is essential for basal transcription in nuclear extracts that contain a more physiological complement of factors (Mittler, G., Kremmer, E., Timmers, H. T., and Meisterernst, M. (2001) EMBO Rep. 2, 808–813; Baek, H. J., Malik, S., Qin, J., and Roeder, R. G. (2002) Mol. Cell. Biol. 22, 2842–2852). Here, mechanistic studies with immobilized DNA templates, purified factors, and factor-depleted HeLa extracts have shown (i) that Mediator enhancement of basal transcription correlates with Mediator-dependent recruitment of pol II and general initiation factors (transcription factor (TF) IIB and TFIIE) to the promoter; (ii) that Mediator and TFIIB, which both interact with pol II, are jointly required for pol II recruitment to the promoter and that TFIIB recruitment is Mediator-dependent, whereas Mediator recruitment is TFIIB-independent; (iii) that a high level of TFIIB can bypass the Mediator requirement for basal transcription and pol II recruitment in nuclear extract, thus indicating a conditional restriction of TFIIB function and a key role of Mediator in overcoming this restriction; and (iv) that an earlier rate-limiting step involves formation of a TFIID-Mediator-promoter complex. These results support a stepwise assembly model, rather than a preformed holoenzyme model, for Mediator-dependent assembly of a basal preinitiation complex and, more important, identify a step involving TFIIB as a key site of action of Mediator. The transcription of eukaryotic genes is mediated by functional interactions of the general transcription machinery with common core promoter (e.g. TATA) elements and further regulated by gene-specific factors bound to distal regulatory elements (1Lee T.I. Young R.A. Annu. Rev. Genet. 2000; 34: 77-137Crossref PubMed Scopus (632) Google Scholar). Studies with purified factors have demonstrated that pol II 4The abbreviations used are: pol II, RNA polymerase II; TF, transcription factor; PIC, preinitiation complex; CTD, C-terminal domain; TAF, TATA box-binding protein-associated factor; TBP, TATA box-binding protein; GTFs, general transcription factors. and cognate initiation factors TFIIA, TFIIB, TFIID, TFIIE, TFIIF, and TFIIH (general transcription machinery) are necessary and sufficient for robust basal (activator-independent) transcription from TATA-containing DNA templates and that the formation of a functional PIC involves the ordered stepwise assembly of these factors (2Roeder R.G. Trends Biochem. Sci. 1996; 21: 327-335Abstract Full Text PDF PubMed Scopus (718) Google Scholar, 3Orphanides G. Lagrange T. Reinberg D. Genes Dev. 1996; 10: 2657-2683Crossref PubMed Scopus (851) Google Scholar). In contrast, activator-dependent transcription from DNA templates was found to require additional cofactors, the most prominent of which is the multisubunit Mediator complex, which is conserved from yeast to human (4Malik S. Roeder R.G. Trends Biochem. Sci. 2005; 30: 256-263Abstract Full Text Full Text PDF PubMed Scopus (314) Google Scholar, 5Myers L.C. Kornberg R.D. Annu. Rev. Biochem. 2000; 69: 729-749Crossref PubMed Scopus (321) Google Scholar). Early demonstrations of its interaction with pol II (6Thompson C.M. Koleske A.J. Chao D.M. Young R.A. Cell. 1993; 73: 1361-1375Abstract Full Text PDF PubMed Scopus (388) Google Scholar, 7Kim Y.J. Bjorklund S. Li Y. Sayre M.H. Kornberg R.D. Cell. 1994; 77: 599-608Abstract Full Text PDF PubMed Scopus (889) Google Scholar), along with later demonstrations of direct interactions with activators (4Malik S. Roeder R.G. Trends Biochem. Sci. 2005; 30: 256-263Abstract Full Text Full Text PDF PubMed Scopus (314) Google Scholar, 8Fondell J.D. Ge H. Roeder R.G. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 8329-8333Crossref PubMed Scopus (464) Google Scholar), led to the notion that Mediator serves as a bridge between DNA-binding regulatory factors and the general transcription machinery to facilitate formation and/or function of the PIC (4Malik S. Roeder R.G. Trends Biochem. Sci. 2005; 30: 256-263Abstract Full Text Full Text PDF PubMed Scopus (314) Google Scholar, 5Myers L.C. Kornberg R.D. Annu. Rev. Biochem. 2000; 69: 729-749Crossref PubMed Scopus (321) Google Scholar). Various biochemical and genetic studies have substantiated this view. Despite its paramount role in activator-dependent transcription, Mediator also has been implicated in basal transcription events by both biochemical and genetic assays. Thus, consistent with the ability of yeast Mediator to enhance basal transcription in a purified system (7Kim Y.J. Bjorklund S. Li Y. Sayre M.H. Kornberg R.D. Cell. 1994; 77: 599-608Abstract Full Text PDF PubMed Scopus (889) Google Scholar), mutations in Mediator subunits were shown to eliminate or reduce basal transcription in crude nuclear extracts (9Koleske A.J. Buratowski S. Nonet M. Young R.A. Cell. 1992; 69: 883-894Abstract Full Text PDF PubMed Scopus (139) Google Scholar, 10Ranish J.A. Yudkovsky N. Hahn S. Genes Dev. 1999; 13: 49-63Crossref PubMed Scopus (206) Google Scholar). This is consistent with genetic studies implicating yeast Mediator in transcription of essentially all genes in yeast (11Holstege F.C. Jennings E.G. Wyrick J.J. Lee T.I. Hengartner C.J. Green M.R. Golub T.R. Lander E.S. Young R.A. Cell. 1998; 95: 717-728Abstract Full Text Full Text PDF PubMed Scopus (1598) Google Scholar). Similarly, human Mediator has also been shown to be essential for basal transcription in nuclear extracts (12Mittler G. Kremmer E. Timmers H.T. Meisterernst M. EMBO Rep. 2001; 2: 808-813Crossref PubMed Scopus (103) Google Scholar, 13Baek H.J. Malik S. Qin J. Roeder R.G. Mol. Cell. Biol. 2002; 22: 2842-2852Crossref PubMed Scopus (109) Google Scholar). As nuclear extracts contain a more natural complement of cellular factors, these assays likely give a more accurate reflection of Mediator requirements in living cells and support the emerging view that Mediator functions as a general transcription factor under more physiological assay conditions. The mechanism(s) by which Mediator facilitates basal transcription remain to be fully understood and could include direct or indirect functions at any of the steps (PIC formation, promoter melting, initiation, promoter clearance, etc.) leading to productive transcription (3Orphanides G. Lagrange T. Reinberg D. Genes Dev. 1996; 10: 2657-2683Crossref PubMed Scopus (851) Google Scholar). The isolation of stable Mediator-pol II (holoenzyme) complexes, initially in yeast (6Thompson C.M. Koleske A.J. Chao D.M. Young R.A. Cell. 1993; 73: 1361-1375Abstract Full Text PDF PubMed Scopus (388) Google Scholar, 7Kim Y.J. Bjorklund S. Li Y. Sayre M.H. Kornberg R.D. Cell. 1994; 77: 599-608Abstract Full Text PDF PubMed Scopus (889) Google Scholar) and later in mammalian cells (4Malik S. Roeder R.G. Trends Biochem. Sci. 2005; 30: 256-263Abstract Full Text Full Text PDF PubMed Scopus (314) Google Scholar, 14Sato S. Tomomori-Sato C. Parmely T.J. Florens L. Zybailov B. Swanson S.K. Banks C.A. Jin J. Cai Y. Washburn M.P. Conaway J.W. Conaway R.C. Mol. Cell. 2004; 14: 685-691Abstract Full Text Full Text PDF PubMed Scopus (250) Google Scholar, 15Malik S. Baek H.J. Wu W. Roeder R.G. Mol. Cell. Biol. 2005; 25: 2117-2129Crossref PubMed Scopus (41) Google Scholar, 16Zhang X. Krutchinsky A. Fukuda A. Chen W. Yamamura S. Chait B.T. Roeder R.G. Mol. Cell. 2005; 19: 89-100Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar), suggested the possibility of Mediator recruitment to, and possible stabilization of, the PIC through Mediator-pol II interactions. A role for Mediator in basal PIC formation or function was also suggested by the ability of yeast Mediator to enhance TFIIH-mediated phosphorylation of the pol II CTD (7Kim Y.J. Bjorklund S. Li Y. Sayre M.H. Kornberg R.D. Cell. 1994; 77: 599-608Abstract Full Text PDF PubMed Scopus (889) Google Scholar) and an associated CTD requirement both for basal transcription in yeast and mammalian nuclear extracts (17Nair D. Kim Y. Myers L.C. J. Biol. Chem. 2005; 280: 33739-33748Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar) and for formation (activator-dependent) of the PIC in yeast nuclear extracts (10Ranish J.A. Yudkovsky N. Hahn S. Genes Dev. 1999; 13: 49-63Crossref PubMed Scopus (206) Google Scholar). Consistent with the demonstration of cooperativity between human Mediator and TAF components of TFIID in basal transcription in nuclear extracts (13Baek H.J. Malik S. Qin J. Roeder R.G. Mol. Cell. Biol. 2002; 22: 2842-2852Crossref PubMed Scopus (109) Google Scholar), human TFIID and Mediator have been reported to act cooperatively in activator-dependent PIC assembly (18Johnson K.M. Wang J. Smallwood A. Arayata C. Carey M. Genes Dev. 2002; 16: 1852-1863Crossref PubMed Scopus (83) Google Scholar). These observations, along with structural studies of the transcription machinery (19Asturias F.J. Curr. Opin. Struct. Biol. 2004; 14: 121-129Crossref PubMed Scopus (38) Google Scholar), are consistent with the view that Mediator interactions with pol II and general initiation factors may facilitate formation and possibly function (initiation, promoter clearance) of the PIC. A recent study has demonstrated activator-independent (basal) functions of Mediator in both PIC formation and re-initiation in yeast extracts (20Reeves W.M. Hahn S. Mol. Cell. Biol. 2003; 23: 349-358Crossref PubMed Scopus (46) Google Scholar). Still unexplained when this study initiated was why basal transcription from DNA templates absolutely requires Mediator in assays with nuclear extracts, but not in assays with purified factors. A direct positive role has been suggested by the recently demonstrated ability of Mediator to stimulate basal transcription (4Malik S. Roeder R.G. Trends Biochem. Sci. 2005; 30: 256-263Abstract Full Text Full Text PDF PubMed Scopus (314) Google Scholar, 7Kim Y.J. Bjorklund S. Li Y. Sayre M.H. Kornberg R.D. Cell. 1994; 77: 599-608Abstract Full Text PDF PubMed Scopus (889) Google Scholar, 21Malik S. Wallberg A.E. Kang Y.K. Roeder R.G. Mol. Cell. Biol. 2002; 22: 5626-5637Crossref PubMed Scopus (83) Google Scholar) or to compensate for limiting amounts of general initiation factors (17Nair D. Kim Y. Myers L.C. J. Biol. Chem. 2005; 280: 33739-33748Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar) in basal transcription assays reconstituted with purified factors. In contrast, an indirect anti-repression effect was suggested from yeast genetic studies in which negative cofactors (NC2 and Not1) that inhibit the general transcription machinery were isolated as suppressors of a mutant Mediator subunit (22Gadbois E.L. Chao D.M. Reese J.C. Green M.R. Young R.A. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3145-3150Crossref PubMed Scopus (54) Google Scholar, 23Lee T.I. Wyrick J.J. Koh S.S. Jennings E.G. Gadbois E.L. Young R.A. Mol. Cell. Biol. 1998; 18: 4455-4462Crossref PubMed Scopus (85) Google Scholar). Although a recent study with yeast nuclear extract failed to show Mediator effects through Not1 and NC2 complexes (24Takagi Y. Kornberg R.D. J. Biol. Chem. 2006; 281: 80-89Abstract Full Text Full Text PDF PubMed Scopus (132) Google Scholar), it did not eliminate possible effects through other known (25Lee T.I. Young R.A. Genes Dev. 1998; 12: 1398-1408Crossref PubMed Scopus (158) Google Scholar, 26Sims III, R.J. Belotserkovskaya R. Reinberg D. Genes Dev. 2004; 18: 2437-2468Crossref PubMed Scopus (571) Google Scholar) or unknown factors that may place constraints on PIC formation or function. Another question of importance in relation to Mediator function and potential targets is whether the poorly characterized basal PIC assembly pathway in nuclear extracts is identical to the pathway established with purified factors. The latter involves TATA element recognition by TBP, generally in the context of TFIID and with possible stabilization by TFIIA, with subsequent sequential binding of TFIIB (to TBP), TFIIF-pol II (to TFIIB), TFIIE (to pol II), and TFIIH (to TFIIE) (2Roeder R.G. Trends Biochem. Sci. 1996; 21: 327-335Abstract Full Text PDF PubMed Scopus (718) Google Scholar, 3Orphanides G. Lagrange T. Reinberg D. Genes Dev. 1996; 10: 2657-2683Crossref PubMed Scopus (851) Google Scholar). In this pathway, pol II recruitment is critically dependent upon prior binding of TFIIB to the TBP/TFIID-promoter complex, whereas TFIID-Mediator and Mediator-pol II interactions (see above) might possibly result in less of a dependence on TFIIB for pol II recruitment in nuclear extracts. Thus, and relevant to this study, there could be some redundancy or cooperativity between TFIIB and Mediator in PIC formation. Related studies of activator-driven PIC assembly in yeast extracts have argued in favor of a holoenzyme model (6Thompson C.M. Koleske A.J. Chao D.M. Young R.A. Cell. 1993; 73: 1361-1375Abstract Full Text PDF PubMed Scopus (388) Google Scholar) involving joint interdependent recruitment of pol II, Mediator, and a subset of general initiation factors (10Ranish J.A. Yudkovsky N. Hahn S. Genes Dev. 1999; 13: 49-63Crossref PubMed Scopus (206) Google Scholar), although cell-based chromatin immunoprecipitation assays have clearly shown that Mediator and pol II are not simultaneously recruited on at least some genes (27Cosma M.P. Panizza S. Nasmyth K. Mol. Cell. 2001; 7: 1213-1220Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar, 28Bhoite L.T. Yu Y. Stillman D.J. Genes Dev. 2001; 15: 2457-2469Crossref PubMed Scopus (109) Google Scholar, 29Park J.M. Werner J. Kim J.M. Lis J.T. Kim Y.J. Mol. Cell. 2001; 8: 9-19Abstract Full Text Full Text PDF PubMed Scopus (117) Google Scholar). In this study, we used immobilized template assays in conjunction with purified factors and factor-depleted nuclear extracts to investigate the PIC assembly pathway and basis of the Mediator requirement for basal transcription in extracts from human cells. Our results demonstrate a Mediator function in basal PIC formation that surprisingly involves facilitated recruitment of TFIIB. They further demonstrate a rate-limiting step in basal transcription that appears to involve formation of a TFIID-Mediator-promoter complex. Purified Factors—Bacterially expressed histidine-tagged TBP was purified through sequential nickel-nitrilotriacetic acid-agarose and heparin-Sepharose chromatography (30Malik S. Roeder R.G. Methods Enzymol. 2003; 364: 257-284Crossref PubMed Scopus (40) Google Scholar). Histidine-tagged TFIIB was purified on nickel-nitrilotriacetic acid-agarose (30Malik S. Roeder R.G. Methods Enzymol. 2003; 364: 257-284Crossref PubMed Scopus (40) Google Scholar). Recombinant TFIIF was reconstituted with individually expressed histidine-tagged RAP74 and untagged RAP30 subunits as described (30Malik S. Roeder R.G. Methods Enzymol. 2003; 364: 257-284Crossref PubMed Scopus (40) Google Scholar). TFIID was purified from a FLAG epitope-tagged TBP-expressing cell line (30Malik S. Roeder R.G. Methods Enzymol. 2003; 364: 257-284Crossref PubMed Scopus (40) Google Scholar). Mediator(f: NUT2) and Mediator(f:TRAP220AB) were isolated from FLAG epitope-tagged MED10/NUT2- and MED1/TRAP220AB-expressing cell lines, respectively (31Malik S. Gu W. Wu W. Qin J. Roeder R.G. Mol. Cell. 2000; 5: 753-760Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar, 32Malik S. Guermah M. Yuan C.X. Wu W. Yamamura S. Roeder R.G. Mol. Cell. Biol. 2004; 24: 8244-8254Crossref PubMed Scopus (81) Google Scholar). Immobilized Templates—Streptavidin-Sepharose beads (Amersham Biosciences) were concentrated by centrifugation and washed twice with 300 μl of Buffer T (10 mm Tris-HCl (pH 7.5), 1 mm EDTA, and 1 m NaCl) supplemented with 0.05% Nonidet P-40 (10Ranish J.A. Yudkovsky N. Hahn S. Genes Dev. 1999; 13: 49-63Crossref PubMed Scopus (206) Google Scholar). The beads were resuspended in Buffer T (0.14 ml of beads/ml of buffer) and incubated with 1.5 pmol of biotinylated template/μl of beads in Buffer T for 30 min at room temperature with constant agitation. The immobilized templates were concentrated by centrifugation and washed three times with 300 μl of Buffer T. The immobilized templates were suspended in block buffer (10 mm Hepes-KOH, pH 7.6, 100 mm potassium glutamate, 10 mm magnesium acetate, 5 mm EGTA 3.5% glycerol, 60 mg/ml casein, and 5 mg/ml polyvinylpyrrolidone) (10 ml/ml of beads) for 15 min at room temperature with constant agitation (10Ranish J.A. Yudkovsky N. Hahn S. Genes Dev. 1999; 13: 49-63Crossref PubMed Scopus (206) Google Scholar). The beads were concentrated by centrifugation, washed three times with transcription buffer (20 mm Hepes-KOH (pH 7.6), 4 mm MgCl2, 60 mm KCl, 0.08 mm EDTA, 8 mm dithiothreitol, 0.4 mg/ml bovine serum albumin, 0.05% Nonidet P-40, and 10% glycerol), and resuspended in BC100 (30Malik S. Roeder R.G. Methods Enzymol. 2003; 364: 257-284Crossref PubMed Scopus (40) Google Scholar) at 0.5 ml/ml. Immobilized templates were prepared fresh before each experiment. In Vitro Transcription with Immobilized Templates—Transcription reactions were carried out in two steps. First, preincubation reactions were set up and contained (in a final volume of 25 μl) 2 μl of immobilized template, 500 ng of sonicated Escherichia coli DNA, 4 μl of HeLa nuclear extract (at ∼10 mg/ml), 20 mm Hepes-KOH (pH 8.2), 11–16% glycerol, 4 mm MgCl2, 60 mm KCl, 8 mm dithiothreitol, 0.4 mg/ml bovine serum albumin, and 20 units of RNasin (Promega). Reactions were incubated at 30 °C for 1 h, and immobilized templates were washed three times with 300 μl of transcription buffer. Second, these immobilized templates were further incubated in 25-μl reactions containing 20 mm Hepes-KOH (pH 8.2), 11–16% glycerol, 4 mm MgCl2, 60 mm KCl, 8 mm dithiothreitol, 0.5 mm each ATP and CTP, 5 μm UTP, 0.1 mm 3′-O-methyl-GTP, 16 μCi (0.6 MBq) of [α-32P]UTP, 0.4 mg/ml bovine serum albumin, and 20 units of RNasin. These reactions were incubated at 30 °C for 1 h, at which time the UTP concentration was increased to 25 μm, and 15 units of RNase T1 were added. After a 30-min incubation at 30 °C, the reactions were extracted with phenol/chloroform in the presence of 150 μl of stop solution (0.4 mm sodium acetate (pH 5.2), 13 mm EDTA, 0.33% SDS, and 0.67 mg/ml yeast tRNA). The aqueous layer was precipitated by ethanol and analyzed by gel electrophoresis followed by autoradiography. Correctly initiated transcripts were quantitated by PhosphorImager analysis using a GE Healthcare STORM 840 system. Analysis of Proteins on Immobilized Templates—For analysis of proteins bound to immobilized templates, scaled-up (100 μl) transcription reactions were used. After incubation of extract with immobilized templates, the templates were concentrated by centrifugation and washed three times with 300 μl of transcription buffer. Templates were resuspended in 50 μl of Buffer 3 (New England Biolabs) with 60 units of EcoRI. After incubation for 30 min at 37 °C with constant agitation, the supernatants were collected on compact reaction columns (USB Corp.). Proteins in the supernatants were concentrated by precipitation with trichloroacetic acid and then redissolved in 1× SDS loading buffer. After boiling, samples were resolved on 4–16% polyacrylamide gels (Invitrogen). Proteins were analyzed by immunoblotting. Time Course Assays—Immobilized DNA templates were prepared as described above with minor modifications for convenience of handling. Streptavidin-coupled M-280 Dynabeads (Dynal) were concentrated by Dynal MPC-S and washed twice with 300 μl of Buffer T supplemented with 0.05% Nonidet P-40. Beads were resuspended in Buffer T at 5 μg/ml and incubated with 10 fmol of biotinylated template/μg of beads in Buffer T for 30 min at room temperature with constant agitation. The immobilized templates were concentrated by MPC-S and washed three times with 300 μl of Buffer T. The immobilized templates were blocked in block buffer (0.5 μl/μg of beads) for 15 min at room temperature with constant agitation. The beads were concentrated by MPC-S, washed three times with transcription buffer, and resuspended in BC100 at 0.1 ml/μg. This immobilized template was incubated with HeLa nuclear extract for time t. After washing the immobilized template twice with transcription buffer, either transcription was initiated by addition of NTPs, or bound proteins were analyzed by immunoblotting. Immunodepletion of Nuclear Extracts—Nuclear extracts depleted of Mediator (referred to as NE(ΔMED)) or TFIID and Mediator (NE(ΔIID/ ΔMED)) were prepared as described (13Baek H.J. Malik S. Qin J. Roeder R.G. Mol. Cell. Biol. 2002; 22: 2842-2852Crossref PubMed Scopus (109) Google Scholar). For preparation of nuclear extract lacking TFIIB (NE(ΔIIB)), 200μl of anti-TFIIB antisera (provided by Dr. Sohail Malik) were purified on 100 μl of protein A-Sepharose (Amersham Biosciences) and then cross-linked with dimethyl pimelimidate (Sigma). HeLa nuclear extract (100 μl) in BC100 was incubated with anti-TFIIB antibody-protein A-Sepharose at 4 °C for 4 h. The supernatant was collected, and the immunoprecipitate was eluted with 100 mm glycine (pH 2.5) after extensive washing with BC100. The supernatant and eluate were analyzed by immunoblotting. Nuclear extract lacking TFIIB, TFIID, and Mediator (NE(ΔIIB/ΔIID/ΔMED)) was similarly prepared from NE(ΔIID/ΔMED). pol II, TFIIB, and TFIIE Are Recruited to a TATA-containing Promoter in a Mediator-dependent Manner—Whereas pol II and general transcription factors suffice for robust basal transcription in purified systems (2Roeder R.G. Trends Biochem. Sci. 1996; 21: 327-335Abstract Full Text PDF PubMed Scopus (718) Google Scholar, 3Orphanides G. Lagrange T. Reinberg D. Genes Dev. 1996; 10: 2657-2683Crossref PubMed Scopus (851) Google Scholar), basal transcription in a crude HeLa nuclear extract-based transcription system requires, in addition, human Mediator, as shown in other studies (12Mittler G. Kremmer E. Timmers H.T. Meisterernst M. EMBO Rep. 2001; 2: 808-813Crossref PubMed Scopus (103) Google Scholar, 13Baek H.J. Malik S. Qin J. Roeder R.G. Mol. Cell. Biol. 2002; 22: 2842-2852Crossref PubMed Scopus (109) Google Scholar). Because PIC assembly is an essential and generally rate-limiting step in the overall transcription reaction (33Dvir A. Conaway J.W. Conaway R.C. Curr. Opin. Genet. Dev. 2001; 11: 209-214Crossref PubMed Scopus (105) Google Scholar, 34Fiedler U. Timmers H.T.M. BioEssays. 2000; 22: 316-326Crossref PubMed Scopus (17) Google Scholar), we first investigated the possibility that Mediator enhances basal transcription in the nuclear extract assay by contributing to PIC assembly. To this end, we employed an immobilized template assay (10Ranish J.A. Yudkovsky N. Hahn S. Genes Dev. 1999; 13: 49-63Crossref PubMed Scopus (206) Google Scholar) that allows, after template incubation with nuclear extract and subsequent washing, assessment of both bound factors (by immunoblotting) and transcription activity (by incubation with nucleoside triphosphates). To avoid the problem of substantial TATA-independent binding of endogenous TFIID and Mediator under basal transcription assay conditions (data not shown) and to assess the requirement for Mediator in PIC formation, we employed NE(ΔIID/ΔMED) supplemented with TBP and variably with highly purified Mediator. The TFIID- and Mediator-depleted extract has been described previously (13Baek H.J. Malik S. Qin J. Roeder R.G. Mol. Cell. Biol. 2002; 22: 2842-2852Crossref PubMed Scopus (109) Google Scholar), and the affinity-purified Mediator(f:TRAP220AB) (32Malik S. Guermah M. Yuan C.X. Wu W. Yamamura S. Roeder R.G. Mol. Cell. Biol. 2004; 24: 8244-8254Crossref PubMed Scopus (81) Google Scholar) and Mediator(f:NUT2) (31Malik S. Gu W. Wu W. Qin J. Roeder R.G. Mol. Cell. 2000; 5: 753-760Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar) preparations used in this study are shown in Fig. 1B. As shown in Fig. 1C, TBP, TFIIB, TFIIE, and pol II, as well as ectopic Mediator, were recruited to the promoter in a TATA-dependent manner (lane 3 versus lane 2). Very significantly, recruitment of TFIIB, TFIIE, and pol II was completely dependent upon the presence of Mediator (lane 4 versus lane 3). Recruitment of complete Mediator, rather than the PC2-CRSP complex that lacks the CDK8 module (31Malik S. Gu W. Wu W. Qin J. Roeder R.G. Mol. Cell. 2000; 5: 753-760Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar), is indicated by recruitment of MED12/TRAP230 along with core subunits MED6, MED17/TRAP80, and MED30/TRAP25. Under the conditions of the assay, TFIIF and TFIIH bound to the immobilized template in the absence of either Mediator (lane 4) or an intact TATA element (lane 2). This likely reflects the nonspecific DNA binding activities of these factors and has precluded assessment in this study of the possible Mediator dependence of their recruitment to the PIC at the TATA element. Nonetheless, the results clearly indicate that, in the nuclear extract context, Mediator plays an important role in recruitment to the basal promoter of key components (minimally TFIIB, TFIIE, and pol II) of the PIC. Mediator-dependent Basal Transcription Activity in Nuclear Extracts Correlates with Mediator-dependent Recruitment of Factors to the PIC—A corresponding analysis of the transcription activity of the PIC assembled on the immobilized template revealed a close correlation with the factor binding results. Thus, basal transcription showed an almost complete (49-fold) dependence upon an intact TATA element (Fig. 1D, lane 3 versus lane 2) and a >5-fold dependence upon Mediator (lane 3 versus lane 4), clearly reflecting the TATA- and Mediator-dependent recruitment of pol II and the GTFs observed in Fig. 1C. However, because these assays contained TBP in place of TFIID for technical reasons (see above) and because TAFs contribute to basal transcription in nuclear extracts (13Baek H.J. Malik S. Qin J. Roeder R.G. Mol. Cell. Biol. 2002; 22: 2842-2852Crossref PubMed Scopus (109) Google Scholar), these results may underestimate the true contribution of Mediator to basal transcription. To confirm and extend the single time point correlation between transcription factor recruitment and transcription activity, we compared the temporal recruitment of transcription factors with the temporal acquisition of transcription activity according to the scheme in Fig. 2A. This analysis employed a Mediator-depleted nuclear extract supplemented with purified Mediator(f:TRAP220AB) to avoid the nonspecific binding activity of endogenous Mediator. As shown in Fig. 2 (B and C), the transcription activity of the immobilized template complex increased linearly (up to 8-fold in 32 min) with the time t of preincubation (prior to washing) with the nuclear extract. As shown in Fig. 2D, the recruitment of TFIIB, TFIIE, pol II, and Mediator showed comparable increases (4–8-fold) over the same time period. In contrast and for reasons (nonspecific DNA binding) described above, TFIIH and TBP/TFIID showed constitutively high binding. The close correlation between the temporal formation of functional PICs (exhibiting transcription activity) and the temporal recruitment of Mediator and key PIC components again supports the hypothesis that Mediator-dependent recruitment of PIC components governs the level of basal transcription in a nuclear extract. Mediator Contributes to Activator-dependent Transcription through Enhanced Recruitment of GTFs—The above experiments investigated how Mediator contributes to basal transcription. If this is attributed, as suggested, to Mediator-dependent recruitment of GTFs such as pol II, TFIIB, and TFIIE, the same molecular mechanism might also be relevant to the previously reported role of Mediator in transcription activation by DNA-binding regulatory factors (4Malik S. Roeder R.G. Trends Biochem. Sci. 2005; 30: 256-263Abstract Full Text Full Text PDF PubMed Scopus (314) Google Scholar). Indeed, previous studies have documented activ" @default.
• W2082616562 created "2016-06-24" @default.
• W2082616562 creator A5002717277 @default.
• W2082616562 creator A5007755251 @default.
• W2082616562 creator A5020447204 @default.
• W2082616562 date "2006-06-01" @default.
• W2082616562 modified "2023-09-26" @default.
• W2082616562 title "Human Mediator Enhances Basal Transcription by Facilitating Recruitment of Transcription Factor IIB during Preinitiation Complex Assembly" @default.
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