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W2139563276 abstract "The TATA box-binding protein (TBP) and TBP-associated factors (TAFIIs) compose the general transcription factor TFIID. The TAFII subunits mediate activated transcription by RNA polymerase II by interacting directly with site-specific transcriptional regulators. TAFIIs also participate in promoter recognition by contacting core promoter elements in the context of TFIID. To further dissect the contribution of individual TAFII subunits to mammalian TFIID function, we employed a vaccinia virus-based protein expression system to study protein-protein interactions and complex assembly. We identified the domains of human (h) TAFII130 required for TAFII-TAFII interactions and formation of a complex with hTBP, hTAFII100, and hTAFII250. Functional analysis of partial TFIID complexes formed in vivo indicated that hTAFII130 was required for transcriptional activation by Sp1 in vitro. DNase I footprinting experiments demonstrated that purified hTBP/hTAFII250 complex reconstituted with or without additional TAFIIs was significantly reduced for TATA box binding (as much as 9-fold) compared with free hTBP. By contrast, hTAFII130 stabilized binding of hTBP to the TATA box, whereas hTAFII100 had little effect. Thus, our biochemical analysis supports the notion that TAFIIs possess distinct functions to regulate the activity of TFIID. The TATA box-binding protein (TBP) and TBP-associated factors (TAFIIs) compose the general transcription factor TFIID. The TAFII subunits mediate activated transcription by RNA polymerase II by interacting directly with site-specific transcriptional regulators. TAFIIs also participate in promoter recognition by contacting core promoter elements in the context of TFIID. To further dissect the contribution of individual TAFII subunits to mammalian TFIID function, we employed a vaccinia virus-based protein expression system to study protein-protein interactions and complex assembly. We identified the domains of human (h) TAFII130 required for TAFII-TAFII interactions and formation of a complex with hTBP, hTAFII100, and hTAFII250. Functional analysis of partial TFIID complexes formed in vivo indicated that hTAFII130 was required for transcriptional activation by Sp1 in vitro. DNase I footprinting experiments demonstrated that purified hTBP/hTAFII250 complex reconstituted with or without additional TAFIIs was significantly reduced for TATA box binding (as much as 9-fold) compared with free hTBP. By contrast, hTAFII130 stabilized binding of hTBP to the TATA box, whereas hTAFII100 had little effect. Thus, our biochemical analysis supports the notion that TAFIIs possess distinct functions to regulate the activity of TFIID. TATA box-binding protein TBP-associated factor hemagglutinin human polyacrylamide gel electrophoresis adenovirus major late promoter Regulation of transcription in eukaryotes requires the participation of a number of transcription factors, many of which exist as multiprotein complexes. The general transcription factor TFIID is one such complex composed of the TATA box-binding protein (TBP)1 and multiple TBP-associated factors (TAFIIs) and is required at many gene promoters to initiate transcription by RNA polymerase II. In the context of TFIID, TAFIIs have been shown to interact with 1) specific transcriptional activators to mediate activation, 2) basal transcription factors, 3) other TAFIIs, and 4) specific DNA sequences, such as the downstream promoter element or gene-specific core promoter sequence, thereby contributing to promoter selectivity (reviewed in Refs. 1Burley S.K. Roeder R.G. Annu. Rev. Biochem. 1996; 65: 769-799Crossref PubMed Scopus (621) Google Scholar, 2Hahn S. Cell. 1998; 95: 579-582Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar, 3Hoffmann A. Oelgeschlager T. Roeder R.G. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 8928-8935Crossref PubMed Scopus (76) Google Scholar, 4Lee T.I. Young R.A. Genes Dev. 1998; 12: 1398-1408Crossref PubMed Scopus (155) Google Scholar, 5Orphanides G. Lagrange T. Reinberg D. Genes Dev. 1996; 10: 2657-2683Crossref PubMed Scopus (843) Google Scholar, 6Roeder R.G. Trends Biochem. Sci. 1996; 21: 327-335Abstract Full Text PDF PubMed Scopus (718) Google Scholar, 7Sauer F. Tjian R. Curr. Opin. Genet. Dev. 1997; 7: 176-181Crossref PubMed Scopus (77) Google Scholar). Recent discovery of a subset of TAFIIs shared by TFIID and the chromatin remodeling complexes PCAF and SAGA (8Grant P.A. Schieltz D. Pray-Grant M.G. Steger D.J. Reese J.C. Yates III, J.R. Workman J.L. Cell. 1998; 94: 45-53Abstract Full Text Full Text PDF PubMed Scopus (385) Google Scholar, 9Ogryzko V.V. Kotani T. Zhang X. Schlitz R.L. Howard T. Yang X.J. Howard B.H. Qin J. Nakatani Y. Cell. 1998; 94: 35-44Abstract Full Text Full Text PDF PubMed Scopus (466) Google Scholar), as well as TBP-free TAFII-containing complex (10Wieczorek E. Brand M. Jacq X. Tora L. Nature. 1998; 393: 187-191Crossref PubMed Scopus (225) Google Scholar) further points to multiple functions of TAFIIs in regulating gene expression in eukaryotes (reviewed in Refs. 11Green M.R. Trends Biochem. Sci. 2000; 25: 59-63Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar, 12Bjorklund S. Almouzni G. Davidson I. Nightingale K.P. Weiss K. Cell. 1999; 96: 759-767Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar, 13Struhl K. Moqtaderi Z. Cell. 1998; 94: 1-4Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar).Although many of the TAFII functions described above have been demonstrated in vitro, the role of TAFIIsin vivo has been controversial. Genetic experiments inDrosophila point to a role for TAFIIs in activated transcription during development (14Zhou J. Zwicker J. Szymanski P. Levine M. Tjian R. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 13483-13488Crossref PubMed Scopus (60) Google Scholar); however, genetic studies in yeast have challenged the essential role of TAFIIs in regulated transcription suggested from the biochemical studies (15Walker S.S. Reese J.C. Apone L.M. Green M.R. Nature. 1996; 383: 185-188Crossref PubMed Scopus (212) Google Scholar, 16Apone L.M. Virbasius C.A. Reese J.C. Green M.R. Genes Dev. 1996; 10: 2368-2380Crossref PubMed Scopus (129) Google Scholar, 17Moqtaderi Z. Bai Y. Poon D. Weil P.A. Struhl K. Nature. 1996; 383: 188-191Crossref PubMed Scopus (250) Google Scholar, 18Walker S.S. Shen W.-C. Reese J.C. Apone L.M. Green M.R. Cell. 1997; 90: 607-614Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar). More recent studies suggest that the requirement for specific TAFIIs may be gene-specific. Indeed, genome-wide expression profiling in yeast demonstrates that 16% of 6000 genes in the yeast genome are affected by the inactivation of a temperature-sensitive allele of yTAFII145, the homolog of the largest metazoan TAFII250 (19Holstege 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 (1591) Google Scholar). By contrast, the effects of mutations in the yTAFII17 gene as well as yTAFII60 and yTAFII61 indicate that mutations in these TAFII genes lead to generalized reduction in RNA polymerase II transcription (20Apone L.M. Virbasius C.A. Holstege F.C. Wang J. Young R.A. Green M.R. Mol. Cell. 1998; 2: 653-661Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar, 21Moqtaderi Z. Keaveney M. Struhl K. Mol. Cell. 1998; 2: 675-682Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 22Michel B. Komarnitsky P. Buratowski S. Mol. Cell. 1998; 2: 663-673Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar, 23Natarajan K. Jackson B.M. Rhee E. Hinnebusch A.G. Mol. Cell. 1998; 2: 683-692Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). Significantly, all three yTAFIIs contain histone-like folds, which may be essential for maintaining the integrity of the TFIID complex.Inactivation of certain yeast and mammalian TAFIIs results in cell cycle phenotypes; for example, cells arrest in G1/S when yTAFII145 or its mammalian homolog TAFII250 is inactivated (15Walker S.S. Reese J.C. Apone L.M. Green M.R. Nature. 1996; 383: 185-188Crossref PubMed Scopus (212) Google Scholar, 24Sekiguchi T. Nohiro Y. Nakamura Y. Hisamoto N. Nishimoto T. Mol. Cell. Biol. 1991; 11: 3317-3325Crossref PubMed Scopus (117) Google Scholar). Examination of the promoters of genes affected by TAFII250 inactivation has demonstrated that responsiveness to TAFII250 resides in both upstream and core promoter sequences, suggesting that TAFIIs can regulate transcription through the core promoter (25Wang E.H. Zou S. Tjian R. Genes Dev. 1997; 11: 2658-2669Crossref PubMed Scopus (78) Google Scholar, 26Suzuki-Yagawa Y. Guermah M. Roeder R.G. Mol. Cell. Bio. 1997; 17: 3284-3294Crossref PubMed Scopus (72) Google Scholar, 27O'Brien T. Tjian R. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 2456-2461Crossref PubMed Scopus (52) Google Scholar). By contrast, promoter mapping studies of yTAFII145-dependent genes has led to the conclusion that yTAFII145 functions to recognize the core promoter (28Shen W.-C. Green M.R. Cell. 1997; 90: 615-624Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar). In the case of yTAFII17, the upstream activating sequence appears to render some genes yTAFII17-dependent (20Apone L.M. Virbasius C.A. Holstege F.C. Wang J. Young R.A. Green M.R. Mol. Cell. 1998; 2: 653-661Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). These studies collectively suggest that TAFIIs have distinct properties and that a subset of TAFIIs may be required for appropriate expression of each gene. The molecular basis for gene-specific requirement for TAFIIs remains to be determined. A gene may be dependent on a subset of TAFIIs for expression because it is regulated by site-specific transcription factors that require certain TAFIIs for activation or repression. On the other hand, some TAFIIs have been shown to contact core promoter directly, suggesting that TAFIIs can modulate the association of TFIID with promoter DNA in a manner dependent on the specific sequence of the gene promoter.To further characterize the function of individual TAFIIs and the relationship among TAFIIs and TBP within the context of TFIID, we have carried out biochemical analyses utilizing the vaccinia virus protein expression system (29Moss B. Elroy-Stein O. Mizukami T. Alexander W.A. Fuerst T.R. Nature. 1990; 348 (912): 919Crossref Scopus (448) Google Scholar). We have investigated TAFII-TAFII interactions between hTAFII250 (30Ruppert S. Wang E.H. Tjian R. Nature. 1993; 362: 175-179Crossref PubMed Scopus (191) Google Scholar, 31Hisatake K. Hasegawa S. Takada R. Nakatani Y. Horikoshi M. Roeder R.G. Nature. 1993; 362: 179-181Crossref PubMed Scopus (149) Google Scholar), hTAFII130 (32Tanese N. Saluja D. Vassallo M.F. Chen J.-L. Admon A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13611-13616Crossref PubMed Scopus (118) Google Scholar, 33Mengus G. May M. Carre L. Chambon P. Davidson I. Genes Dev. 1997; 11: 1381-1395Crossref PubMed Scopus (132) Google Scholar), and hTAFII100 (32Tanese N. Saluja D. Vassallo M.F. Chen J.-L. Admon A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13611-13616Crossref PubMed Scopus (118) Google Scholar, 34Dubrovskaya V. Lavigne A.-C. Davidson I. Acker J. Staub A. Tora L. EMBO J. 1996; 15: 3702-3712Crossref PubMed Scopus (78) Google Scholar, 35Tao Y. Guermah M. Martinez E. Oelgeschlager T. Hasegawa S. Takada R. Yamamoto T. Horikoshi M. Roeder R.G. J. Biol. Chem. 1997; 272: 6714-6721Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar) in pairwise combinations as well as in the partial complexes containing two or more subunits. The vaccinia virus protein expression system offers a number of advantages: 1) The recombinant proteins are expressed in mammalian cells and thus receive appropriate post-translational modifications; 2) the vaccinia virus/T7 RNA polymerase hybrid system permits coordinated expression of multiple genes by the T7 promoter (36Fuerst T.R. Earl P.L. Moss B. Mol. Cell. Biol. 1987; 7: 2538-2544Crossref PubMed Scopus (331) Google Scholar); 3) transient transfection of T7 promoter-regulated genes following infection with a virus expressing the T7 RNA polymerase leads to robust expression permitting the analysis of protein-protein interactions and complex assembly; and 4) coinfection with recombinant viruses carrying T7 promoter-regulated genes facilitates large scale production of proteins in a stable complex that can be purified and used in biochemical analyses. The transient transfection protocol allowed us to test a series of mutant constructs of hTAFII130 for TAFII-TAFII interactions and their ability to assemble into a partial complex with other subunits without the need to make recombinant viruses for each mutant. The recombinant viruses generated were used for purification of dimeric, trimeric, and tetrameric complexes.Using these vaccinia virus-based assays, we examined the assembly among TBP and different TAFII subunits and mapped the “surfaces” required for these interactions. We show the formation of TFIID complexes containing two, three, or four subunits and their role in transcriptional activation and TATA box binding. The functional analysis of individual TAFII subunits both separately and in the context of the partial TFIID complexes has permitted us to assess the role of each subunit in TFIID function. Regulation of transcription in eukaryotes requires the participation of a number of transcription factors, many of which exist as multiprotein complexes. The general transcription factor TFIID is one such complex composed of the TATA box-binding protein (TBP)1 and multiple TBP-associated factors (TAFIIs) and is required at many gene promoters to initiate transcription by RNA polymerase II. In the context of TFIID, TAFIIs have been shown to interact with 1) specific transcriptional activators to mediate activation, 2) basal transcription factors, 3) other TAFIIs, and 4) specific DNA sequences, such as the downstream promoter element or gene-specific core promoter sequence, thereby contributing to promoter selectivity (reviewed in Refs. 1Burley S.K. Roeder R.G. Annu. Rev. Biochem. 1996; 65: 769-799Crossref PubMed Scopus (621) Google Scholar, 2Hahn S. Cell. 1998; 95: 579-582Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar, 3Hoffmann A. Oelgeschlager T. Roeder R.G. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 8928-8935Crossref PubMed Scopus (76) Google Scholar, 4Lee T.I. Young R.A. Genes Dev. 1998; 12: 1398-1408Crossref PubMed Scopus (155) Google Scholar, 5Orphanides G. Lagrange T. Reinberg D. Genes Dev. 1996; 10: 2657-2683Crossref PubMed Scopus (843) Google Scholar, 6Roeder R.G. Trends Biochem. Sci. 1996; 21: 327-335Abstract Full Text PDF PubMed Scopus (718) Google Scholar, 7Sauer F. Tjian R. Curr. Opin. Genet. Dev. 1997; 7: 176-181Crossref PubMed Scopus (77) Google Scholar). Recent discovery of a subset of TAFIIs shared by TFIID and the chromatin remodeling complexes PCAF and SAGA (8Grant P.A. Schieltz D. Pray-Grant M.G. Steger D.J. Reese J.C. Yates III, J.R. Workman J.L. Cell. 1998; 94: 45-53Abstract Full Text Full Text PDF PubMed Scopus (385) Google Scholar, 9Ogryzko V.V. Kotani T. Zhang X. Schlitz R.L. Howard T. Yang X.J. Howard B.H. Qin J. Nakatani Y. Cell. 1998; 94: 35-44Abstract Full Text Full Text PDF PubMed Scopus (466) Google Scholar), as well as TBP-free TAFII-containing complex (10Wieczorek E. Brand M. Jacq X. Tora L. Nature. 1998; 393: 187-191Crossref PubMed Scopus (225) Google Scholar) further points to multiple functions of TAFIIs in regulating gene expression in eukaryotes (reviewed in Refs. 11Green M.R. Trends Biochem. Sci. 2000; 25: 59-63Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar, 12Bjorklund S. Almouzni G. Davidson I. Nightingale K.P. Weiss K. Cell. 1999; 96: 759-767Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar, 13Struhl K. Moqtaderi Z. Cell. 1998; 94: 1-4Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar). Although many of the TAFII functions described above have been demonstrated in vitro, the role of TAFIIsin vivo has been controversial. Genetic experiments inDrosophila point to a role for TAFIIs in activated transcription during development (14Zhou J. Zwicker J. Szymanski P. Levine M. Tjian R. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 13483-13488Crossref PubMed Scopus (60) Google Scholar); however, genetic studies in yeast have challenged the essential role of TAFIIs in regulated transcription suggested from the biochemical studies (15Walker S.S. Reese J.C. Apone L.M. Green M.R. Nature. 1996; 383: 185-188Crossref PubMed Scopus (212) Google Scholar, 16Apone L.M. Virbasius C.A. Reese J.C. Green M.R. Genes Dev. 1996; 10: 2368-2380Crossref PubMed Scopus (129) Google Scholar, 17Moqtaderi Z. Bai Y. Poon D. Weil P.A. Struhl K. Nature. 1996; 383: 188-191Crossref PubMed Scopus (250) Google Scholar, 18Walker S.S. Shen W.-C. Reese J.C. Apone L.M. Green M.R. Cell. 1997; 90: 607-614Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar). More recent studies suggest that the requirement for specific TAFIIs may be gene-specific. Indeed, genome-wide expression profiling in yeast demonstrates that 16% of 6000 genes in the yeast genome are affected by the inactivation of a temperature-sensitive allele of yTAFII145, the homolog of the largest metazoan TAFII250 (19Holstege 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 (1591) Google Scholar). By contrast, the effects of mutations in the yTAFII17 gene as well as yTAFII60 and yTAFII61 indicate that mutations in these TAFII genes lead to generalized reduction in RNA polymerase II transcription (20Apone L.M. Virbasius C.A. Holstege F.C. Wang J. Young R.A. Green M.R. Mol. Cell. 1998; 2: 653-661Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar, 21Moqtaderi Z. Keaveney M. Struhl K. Mol. Cell. 1998; 2: 675-682Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 22Michel B. Komarnitsky P. Buratowski S. Mol. Cell. 1998; 2: 663-673Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar, 23Natarajan K. Jackson B.M. Rhee E. Hinnebusch A.G. Mol. Cell. 1998; 2: 683-692Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). Significantly, all three yTAFIIs contain histone-like folds, which may be essential for maintaining the integrity of the TFIID complex. Inactivation of certain yeast and mammalian TAFIIs results in cell cycle phenotypes; for example, cells arrest in G1/S when yTAFII145 or its mammalian homolog TAFII250 is inactivated (15Walker S.S. Reese J.C. Apone L.M. Green M.R. Nature. 1996; 383: 185-188Crossref PubMed Scopus (212) Google Scholar, 24Sekiguchi T. Nohiro Y. Nakamura Y. Hisamoto N. Nishimoto T. Mol. Cell. Biol. 1991; 11: 3317-3325Crossref PubMed Scopus (117) Google Scholar). Examination of the promoters of genes affected by TAFII250 inactivation has demonstrated that responsiveness to TAFII250 resides in both upstream and core promoter sequences, suggesting that TAFIIs can regulate transcription through the core promoter (25Wang E.H. Zou S. Tjian R. Genes Dev. 1997; 11: 2658-2669Crossref PubMed Scopus (78) Google Scholar, 26Suzuki-Yagawa Y. Guermah M. Roeder R.G. Mol. Cell. Bio. 1997; 17: 3284-3294Crossref PubMed Scopus (72) Google Scholar, 27O'Brien T. Tjian R. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 2456-2461Crossref PubMed Scopus (52) Google Scholar). By contrast, promoter mapping studies of yTAFII145-dependent genes has led to the conclusion that yTAFII145 functions to recognize the core promoter (28Shen W.-C. Green M.R. Cell. 1997; 90: 615-624Abstract Full Text Full Text PDF PubMed Scopus (165) Google Scholar). In the case of yTAFII17, the upstream activating sequence appears to render some genes yTAFII17-dependent (20Apone L.M. Virbasius C.A. Holstege F.C. Wang J. Young R.A. Green M.R. Mol. Cell. 1998; 2: 653-661Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). These studies collectively suggest that TAFIIs have distinct properties and that a subset of TAFIIs may be required for appropriate expression of each gene. The molecular basis for gene-specific requirement for TAFIIs remains to be determined. A gene may be dependent on a subset of TAFIIs for expression because it is regulated by site-specific transcription factors that require certain TAFIIs for activation or repression. On the other hand, some TAFIIs have been shown to contact core promoter directly, suggesting that TAFIIs can modulate the association of TFIID with promoter DNA in a manner dependent on the specific sequence of the gene promoter. To further characterize the function of individual TAFIIs and the relationship among TAFIIs and TBP within the context of TFIID, we have carried out biochemical analyses utilizing the vaccinia virus protein expression system (29Moss B. Elroy-Stein O. Mizukami T. Alexander W.A. Fuerst T.R. Nature. 1990; 348 (912): 919Crossref Scopus (448) Google Scholar). We have investigated TAFII-TAFII interactions between hTAFII250 (30Ruppert S. Wang E.H. Tjian R. Nature. 1993; 362: 175-179Crossref PubMed Scopus (191) Google Scholar, 31Hisatake K. Hasegawa S. Takada R. Nakatani Y. Horikoshi M. Roeder R.G. Nature. 1993; 362: 179-181Crossref PubMed Scopus (149) Google Scholar), hTAFII130 (32Tanese N. Saluja D. Vassallo M.F. Chen J.-L. Admon A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13611-13616Crossref PubMed Scopus (118) Google Scholar, 33Mengus G. May M. Carre L. Chambon P. Davidson I. Genes Dev. 1997; 11: 1381-1395Crossref PubMed Scopus (132) Google Scholar), and hTAFII100 (32Tanese N. Saluja D. Vassallo M.F. Chen J.-L. Admon A. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 13611-13616Crossref PubMed Scopus (118) Google Scholar, 34Dubrovskaya V. Lavigne A.-C. Davidson I. Acker J. Staub A. Tora L. EMBO J. 1996; 15: 3702-3712Crossref PubMed Scopus (78) Google Scholar, 35Tao Y. Guermah M. Martinez E. Oelgeschlager T. Hasegawa S. Takada R. Yamamoto T. Horikoshi M. Roeder R.G. J. Biol. Chem. 1997; 272: 6714-6721Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar) in pairwise combinations as well as in the partial complexes containing two or more subunits. The vaccinia virus protein expression system offers a number of advantages: 1) The recombinant proteins are expressed in mammalian cells and thus receive appropriate post-translational modifications; 2) the vaccinia virus/T7 RNA polymerase hybrid system permits coordinated expression of multiple genes by the T7 promoter (36Fuerst T.R. Earl P.L. Moss B. Mol. Cell. Biol. 1987; 7: 2538-2544Crossref PubMed Scopus (331) Google Scholar); 3) transient transfection of T7 promoter-regulated genes following infection with a virus expressing the T7 RNA polymerase leads to robust expression permitting the analysis of protein-protein interactions and complex assembly; and 4) coinfection with recombinant viruses carrying T7 promoter-regulated genes facilitates large scale production of proteins in a stable complex that can be purified and used in biochemical analyses. The transient transfection protocol allowed us to test a series of mutant constructs of hTAFII130 for TAFII-TAFII interactions and their ability to assemble into a partial complex with other subunits without the need to make recombinant viruses for each mutant. The recombinant viruses generated were used for purification of dimeric, trimeric, and tetrameric complexes. Using these vaccinia virus-based assays, we examined the assembly among TBP and different TAFII subunits and mapped the “surfaces” required for these interactions. We show the formation of TFIID complexes containing two, three, or four subunits and their role in transcriptional activation and TATA box binding. The functional analysis of individual TAFII subunits both separately and in the context of the partial TFIID complexes has permitted us to assess the role of each subunit in TFIID function. We acknowledge the technical assistance of Kelly Vogel and Amy Kun. We are grateful to the following researchers for providing valuable reagents: Bernie Moss (National Institutes of Health) for vaccinia viral vectors, Stewart Shuman (Sloan-Kettering Memorial Institute) for recombinant vaccinia virus, Edith Wang (University of Washington) for antisera against TAFIIs and insect cell extract overexpressing recombinant hTAFII250, Carla Inouye (University of California, Berkeley) for recombinant human TFIIA protein, Nouria Hernandez (Cold Spring Harbor Laboratory) for SL39 antiserum, Steve Smale (University of California, Los Angeles) for cDNA and antiserum against CIF150, Angus Wilson (New York University School of Medicine) for purified POU DNA binding domain of Oct 1, and David King (University of California, Berkeley) for peptides. HeLa cells used to prepare the nuclear extracts were grown and harvested at the National Cell Culture Center (Minneapolis, MN), whose service is hereby acknowledged. We thank Michael Garabedian and Angus Wilson for valuable suggestions and comments on the manuscript." @default.
W2139563276 created "2016-06-24" @default.
W2139563276 creator A5000153057 @default.
W2139563276 creator A5090371551 @default.
W2139563276 date "2000-09-01" @default.
W2139563276 modified "2023-09-27" @default.
W2139563276 title "Assembly of Partial TFIID Complexes in Mammalian Cells Reveals Distinct Activities Associated with Individual TATA Box-binding Protein-associated Factors" @default.
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