Interactions of a DNA-bound Transcriptional Activator with the TBP-TFIIA-TFIIB-Promoter Quaternary Complex

Site-specific protein-DNA photo-cross-linking was used to show that, when bound to its cognate site at various distances upstream of the TATA element, the chimeric transcriptional activator GAL4-VP16 can physically interact with a TATA box-binding protein (TBP)- transcription factor IIA (TFIIA)-TFIIB complex assembled on the TATA element. This result implies DNA bending and looping of promoter DNA as a result of the physical interaction between GAL4-VP16 and an interface of the TBP-TFIIA-TFIIB complex. This protein-protein interaction on promoter DNA minimally requires the presence of one GAL4 binding site and the formation of a quaternary complex containing TBP, TFIIB, and TFIIA on the TATA element. Notably, the topology of the TBP-TFIIA-TFIIB-promoter complex is not altered significantly by the interaction with DNA-bound activators. We also show that the ability of GAL4-VP16 to activate transcription through a single GAL4 binding site varies according to its precise location and orientation relative to the TATA element and that it can approach the efficiency obtained with multiple binding sites. Taken together, our results indicate that the spatial positioning of the DNA-bound activation domain is important for efficient activation, possibly by maximizing its interactions with the transcriptional machinery including the TBP-TFIIA-TFIIB-promoter quaternary complex. Site-specific protein-DNA photo-cross-linking was used to show that, when bound to its cognate site at various distances upstream of the TATA element, the chimeric transcriptional activator GAL4-VP16 can physically interact with a TATA box-binding protein (TBP)- transcription factor IIA (TFIIA)-TFIIB complex assembled on the TATA element. This result implies DNA bending and looping of promoter DNA as a result of the physical interaction between GAL4-VP16 and an interface of the TBP-TFIIA-TFIIB complex. This protein-protein interaction on promoter DNA minimally requires the presence of one GAL4 binding site and the formation of a quaternary complex containing TBP, TFIIB, and TFIIA on the TATA element. Notably, the topology of the TBP-TFIIA-TFIIB-promoter complex is not altered significantly by the interaction with DNA-bound activators. We also show that the ability of GAL4-VP16 to activate transcription through a single GAL4 binding site varies according to its precise location and orientation relative to the TATA element and that it can approach the efficiency obtained with multiple binding sites. Taken together, our results indicate that the spatial positioning of the DNA-bound activation domain is important for efficient activation, possibly by maximizing its interactions with the transcriptional machinery including the TBP-TFIIA-TFIIB-promoter quaternary complex. RNA polymerase II adenovirus-2 major late RNA polymerase II-associated protein TATA box-binding protein transcription factor Most models of transcriptional activation imply a physical interaction of DNA-bound transcriptional activators and the transcription machinery assembled on core promoters (1Ptashne M. Gann A. Nature. 1997; 386: 569-577Google Scholar, 2Sauer F. Tjian R. Curr. Opin. Genet. Dev. 1997; 7: 176-181Google Scholar, 3Berk A.J. Curr. Opin. Cell Biol. 1999; 11: 330-335Google Scholar). This contact between the activation domain and components of the transcription machinery has been attributed diverse functions including: (i) the recruitment of key transcription factors (e.g. general transcription factors or co-activators) at the promoter, (ii) the stimulation of enzymatic activities involved in the transcription reaction (e.g. promoter melting, phosphorylation, initiation of RNA chain synthesis), and (iii) the relief of transcriptional blockades induced by various types of repressors including nucleosomes. In support of this view of transcriptional activation, a number of protein-protein interactions between various activation domains and members of the RNA polymerase II (RNAPII)1 transcription machinery have been characterized in solution and found to be important in mediating transcriptional activation (1Ptashne M. Gann A. Nature. 1997; 386: 569-577Google Scholar, 2Sauer F. Tjian R. Curr. Opin. Genet. Dev. 1997; 7: 176-181Google Scholar, 3Berk A.J. Curr. Opin. Cell Biol. 1999; 11: 330-335Google Scholar). However, little is known about the formation of these protein-protein interactions when the interacting partners are bound to promoter DNA.Ultimately, transcriptional activators regulate the activity of the basal RNAPII transcription machinery. The transcription reaction is a multi-step process in which a preinitiation complex containing TBP, TFIIB, TFIIE, TFIIF, TFIIH, and RNAPII is first assembled onto promoter DNA (4Orphanides G. Lagrange T. Reinberg D. Genes Dev. 1996; 10: 2657-2683Google Scholar, 5Hampsey M. Microbiol. Mol. Biol. Rev. 1998; 62: 465-503Google Scholar). TBP recognizes and binds the TATA element, inducing a bend of about 80° in the DNA helix (6Kim J.L. Nikolov D.B. Burley S.K. Nature. 1993; 365: 520-527Google Scholar, 7Kim Y. Geiger J.H. Hahn S. Sigler P.B. Nature. 1993; 365: 512-520Google Scholar). TFIIB binds to and stabilizes the TBP-promoter complex (8Maldonado E. Ha I. Cortes P. Weis L. Reinberg D. Mol. Cell. Biol. 1990; 10: 6335-6347Google Scholar). TFIIF, a factor composed of two subunits called RAP74 and RAP30, binds tightly to RNAPII (9Flores O. Lu H. Killeen M. Greenblatt J. Burton Z.F. Reinberg D. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 9999-10003Google Scholar, 10Conaway R.C. Garrett K.P. Hanley J.P. Conaway J.W. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 6205-6209Google Scholar), recruits the enzyme to the TBP-TFIIB-promoter complex, and induces an isomerization of the preinitiation complex that includes wrapping of promoter DNA around RNAPII (11Forget D. Robert F. Grondin G. Burton Z.F. Greenblatt J. Coulombe B. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 7150-7155Google Scholar, 12Robert F. Douziech M. Forget D. Egly J.M. Greenblatt J. Burton Z.F. Coulombe B. Mol. Cell. 1998; 2: 341-351Google Scholar). TFIIE, a factor also composed of two subunits called TFIIEα and TFIIEβ, stabilizes the preinitiation complex (13Robert F. Forget D. Li J. Greenblatt J. Coulombe B. J. Biol. Chem. 1996; 271: 8517-8520Google Scholar) and is involved in the melting of promoter DNA at the transcription initiation site through an ATP-independent mechanism (14Flores O. Lu H. Reinberg D. J. Biol. Chem. 1992; 267: 2786-2793Google Scholar, 15Holstege F.C. Tantin D. Carey M. van der Vliet P.C. Timmers H.T. EMBO J. 1995; 14: 810-819Google Scholar). TFIIH, a nine-subunit factor that has both kinase and helicase activities, mediates the ATP-dependent melting of promoter DNA and is involved in the transition between the initiation and elongation states of the complex (16Schaeffer L. Roy R. Humbert S. Moncollin V. Vermeulen W. Hoeijmakers J.H. Chambon P. Egly J.M. Science. 1993; 260: 58-63Google Scholar, 17Drapkin R. Reardon J.T. Ansari A. Huang J.C. Zawel L. Ahn K. Sancar A. Reinberg D. Nature. 1994; 368: 769-772Google Scholar, 18Lu H. Flores O. Weinmann R. Reinberg D. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 10004-10008Google Scholar, 19Serizawa H. Conaway R.C. Conaway J.W. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 7476-7480Google Scholar, 20Dvir A. Conaway R.C. Conaway J.W. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 9006-9010Google Scholar, 21Yan Q. Moreland R.J. Conaway J.W. Conaway R.C. J. Biol. Chem. 1999; 274: 35668-35675Google Scholar) (also see Ref. 22Coulombe B. Burton Z.F. Microbiol. Mol. Biol. Rev. 1999; 63: 457-478Google Scholar for a review). The initiation of chain synthesis proceeds through a cycle of abortive initiation events during which RNAPII synthesizes short 2–10-nucleotide transcripts before escaping the promoter and entering a productive elongation mode (23Holstege F.C. Fiedler U. Timmers H.T. EMBO J. 1997; 16: 7468-7480Google Scholar). The activity of elongating RNAPII is modulated through the action of a number of elongation factors (24Conaway J.W. Shilatifard A. Dvir A. Conaway R.C. Trends Biochem. Sci. 2000; 25: 375-380Google Scholar, 25Shilatifard A. FASEB J. 1998; 12: 1437-1446Google Scholar).The acidic activator GAL4-VP16 is a chimeric polypeptide composed of the DNA-binding domain of the yeast GAL4 protein (amino acids 1–147) and the transcriptional acidic activation domain of the viral protein VP16 (amino acids 412–490) (26Sadowski I. Ma J. Triezenberg S. Ptashne M. Nature. 1988; 335: 563-564Google Scholar). When GAL4 DNA-binding sites are located upstream of the TATA box of a promoter, this activator has been shown to stimulate transcription in a synergistic manner both in vivo and in vitro (see Ref. 27Lin Y.S. Carey M.F. Ptashne M. Green M.R. Cell. 1988; 54: 659-664Google Scholar for example). GAL4-VP16 can act on various steps of the transcription reaction, probably via its many interactions with components of the basal transcriptional machinery. These include interactions with TBP (28Stringer K.F. Ingles C.J. Greenblatt J. Nature. 1990; 345: 783-786Google Scholar, 29Ingles C.J. Shales M. Cress W.D. Triezenberg S.J. Greenblatt J. Nature. 1991; 351: 588-590Google Scholar), TFIIB (30Lin Y.S. Ha I. Maldonado E. Reinberg D. Green M.R. Nature. 1991; 353: 569-571Google Scholar,31Lin Y.S. Green M.R. Cell. 1991; 64: 971-981Google Scholar), TFIIA (32Kobayashi N. Boyer T.G. Berk A.J. Mol. Cell. Biol. 1995; 15: 6465-6473Google Scholar), TFIIH (33Xiao H. Pearson A. Coulombe B. Truant R. Zhang S. Regier J.L. Triezenberg S.J. Reinberg D. Flores O. Ingles C.J. Greenblatt J. Mol. Cell. Biol. 1994; 14: 7013-7024Google Scholar), and hTAFII32 (34Klemm R.D. Goodrich J.A. Zhou S. Tjian R. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 5788-5792Google Scholar). GAL4-VP16 was shown to stimulate three distinct events during the formation of the preinitiation complex: (i) formation of the TFIID-TFIIA-DNA complex (34Klemm R.D. Goodrich J.A. Zhou S. Tjian R. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 5788-5792Google Scholar); (ii) TFIIB recruitment (35Wang W. Gralla J.D. Carey M. Genes Dev. 1992; 6: 1716-1727Google Scholar); and, (iii) RAP30, TFIIEα, and RNAPII recruitment (36Choy B. Green M.R. Nature. 1993; 366: 531-536Google Scholar). A number of reports indicate that GAL4-VP16 can also act on open complex formation (37Wang W. Carey M. Gralla J.D. Science. 1992; 255: 450-453Google Scholar), transcript elongation (38Blau J. Xiao H. McCracken S. O'Hare P. Greenblatt J. Bentley D. Mol. Cell. Biol. 1996; 16: 2044-2055Google Scholar), and reinitiation (39Zawel L. Kumar K.P. Reinberg D. Genes Dev. 1995; 9: 1479-1490Google Scholar, 40Yudkovsky N. Ranish J.A. Hahn S. Nature. 2000; 408: 225-229Google Scholar).Using site-specific protein-DNA photo-cross-linking, we have analyzed the physical interaction between GAL4-VP16 molecules bound to promoter sites various distances from the TATA element and a complex containing TBP, TFIIB, and TFIIA assembled onto the TATA element. Using templates with either one or five GAL4 DNA-binding sites, we show that DNA-bound GAL4-VP16 does indeed cross-link to many positions in the vicinity of the TATA element where a TBP-TFIIA-TFIIB complex is assembled. Interaction with DNA-bound GAL4-VP16 does not significantly modify the conformation of the TBP-TFIIA-TFIIB-promoter complex. We also show that a GAL4-VP16 dimer bound to one GAL4 binding site can stimulate transcription with an efficiency approaching that of GAL4-VP16 dimers bound to five sites if the single dimer is properly positioned upstream of the TATA box. Most models of transcriptional activation imply a physical interaction of DNA-bound transcriptional activators and the transcription machinery assembled on core promoters (1Ptashne M. Gann A. Nature. 1997; 386: 569-577Google Scholar, 2Sauer F. Tjian R. Curr. Opin. Genet. Dev. 1997; 7: 176-181Google Scholar, 3Berk A.J. Curr. Opin. Cell Biol. 1999; 11: 330-335Google Scholar). This contact between the activation domain and components of the transcription machinery has been attributed diverse functions including: (i) the recruitment of key transcription factors (e.g. general transcription factors or co-activators) at the promoter, (ii) the stimulation of enzymatic activities involved in the transcription reaction (e.g. promoter melting, phosphorylation, initiation of RNA chain synthesis), and (iii) the relief of transcriptional blockades induced by various types of repressors including nucleosomes. In support of this view of transcriptional activation, a number of protein-protein interactions between various activation domains and members of the RNA polymerase II (RNAPII)1 transcription machinery have been characterized in solution and found to be important in mediating transcriptional activation (1Ptashne M. Gann A. Nature. 1997; 386: 569-577Google Scholar, 2Sauer F. Tjian R. Curr. Opin. Genet. Dev. 1997; 7: 176-181Google Scholar, 3Berk A.J. Curr. Opin. Cell Biol. 1999; 11: 330-335Google Scholar). However, little is known about the formation of these protein-protein interactions when the interacting partners are bound to promoter DNA. Ultimately, transcriptional activators regulate the activity of the basal RNAPII transcription machinery. The transcription reaction is a multi-step process in which a preinitiation complex containing TBP, TFIIB, TFIIE, TFIIF, TFIIH, and RNAPII is first assembled onto promoter DNA (4Orphanides G. Lagrange T. Reinberg D. Genes Dev. 1996; 10: 2657-2683Google Scholar, 5Hampsey M. Microbiol. Mol. Biol. Rev. 1998; 62: 465-503Google Scholar). TBP recognizes and binds the TATA element, inducing a bend of about 80° in the DNA helix (6Kim J.L. Nikolov D.B. Burley S.K. Nature. 1993; 365: 520-527Google Scholar, 7Kim Y. Geiger J.H. Hahn S. Sigler P.B. Nature. 1993; 365: 512-520Google Scholar). TFIIB binds to and stabilizes the TBP-promoter complex (8Maldonado E. Ha I. Cortes P. Weis L. Reinberg D. Mol. Cell. Biol. 1990; 10: 6335-6347Google Scholar). TFIIF, a factor composed of two subunits called RAP74 and RAP30, binds tightly to RNAPII (9Flores O. Lu H. Killeen M. Greenblatt J. Burton Z.F. Reinberg D. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 9999-10003Google Scholar, 10Conaway R.C. Garrett K.P. Hanley J.P. Conaway J.W. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 6205-6209Google Scholar), recruits the enzyme to the TBP-TFIIB-promoter complex, and induces an isomerization of the preinitiation complex that includes wrapping of promoter DNA around RNAPII (11Forget D. Robert F. Grondin G. Burton Z.F. Greenblatt J. Coulombe B. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 7150-7155Google Scholar, 12Robert F. Douziech M. Forget D. Egly J.M. Greenblatt J. Burton Z.F. Coulombe B. Mol. Cell. 1998; 2: 341-351Google Scholar). TFIIE, a factor also composed of two subunits called TFIIEα and TFIIEβ, stabilizes the preinitiation complex (13Robert F. Forget D. Li J. Greenblatt J. Coulombe B. J. Biol. Chem. 1996; 271: 8517-8520Google Scholar) and is involved in the melting of promoter DNA at the transcription initiation site through an ATP-independent mechanism (14Flores O. Lu H. Reinberg D. J. Biol. Chem. 1992; 267: 2786-2793Google Scholar, 15Holstege F.C. Tantin D. Carey M. van der Vliet P.C. Timmers H.T. EMBO J. 1995; 14: 810-819Google Scholar). TFIIH, a nine-subunit factor that has both kinase and helicase activities, mediates the ATP-dependent melting of promoter DNA and is involved in the transition between the initiation and elongation states of the complex (16Schaeffer L. Roy R. Humbert S. Moncollin V. Vermeulen W. Hoeijmakers J.H. Chambon P. Egly J.M. Science. 1993; 260: 58-63Google Scholar, 17Drapkin R. Reardon J.T. Ansari A. Huang J.C. Zawel L. Ahn K. Sancar A. Reinberg D. Nature. 1994; 368: 769-772Google Scholar, 18Lu H. Flores O. Weinmann R. Reinberg D. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 10004-10008Google Scholar, 19Serizawa H. Conaway R.C. Conaway J.W. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 7476-7480Google Scholar, 20Dvir A. Conaway R.C. Conaway J.W. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 9006-9010Google Scholar, 21Yan Q. Moreland R.J. Conaway J.W. Conaway R.C. J. Biol. Chem. 1999; 274: 35668-35675Google Scholar) (also see Ref. 22Coulombe B. Burton Z.F. Microbiol. Mol. Biol. Rev. 1999; 63: 457-478Google Scholar for a review). The initiation of chain synthesis proceeds through a cycle of abortive initiation events during which RNAPII synthesizes short 2–10-nucleotide transcripts before escaping the promoter and entering a productive elongation mode (23Holstege F.C. Fiedler U. Timmers H.T. EMBO J. 1997; 16: 7468-7480Google Scholar). The activity of elongating RNAPII is modulated through the action of a number of elongation factors (24Conaway J.W. Shilatifard A. Dvir A. Conaway R.C. Trends Biochem. Sci. 2000; 25: 375-380Google Scholar, 25Shilatifard A. FASEB J. 1998; 12: 1437-1446Google Scholar). The acidic activator GAL4-VP16 is a chimeric polypeptide composed of the DNA-binding domain of the yeast GAL4 protein (amino acids 1–147) and the transcriptional acidic activation domain of the viral protein VP16 (amino acids 412–490) (26Sadowski I. Ma J. Triezenberg S. Ptashne M. Nature. 1988; 335: 563-564Google Scholar). When GAL4 DNA-binding sites are located upstream of the TATA box of a promoter, this activator has been shown to stimulate transcription in a synergistic manner both in vivo and in vitro (see Ref. 27Lin Y.S. Carey M.F. Ptashne M. Green M.R. Cell. 1988; 54: 659-664Google Scholar for example). GAL4-VP16 can act on various steps of the transcription reaction, probably via its many interactions with components of the basal transcriptional machinery. These include interactions with TBP (28Stringer K.F. Ingles C.J. Greenblatt J. Nature. 1990; 345: 783-786Google Scholar, 29Ingles C.J. Shales M. Cress W.D. Triezenberg S.J. Greenblatt J. Nature. 1991; 351: 588-590Google Scholar), TFIIB (30Lin Y.S. Ha I. Maldonado E. Reinberg D. Green M.R. Nature. 1991; 353: 569-571Google Scholar,31Lin Y.S. Green M.R. Cell. 1991; 64: 971-981Google Scholar), TFIIA (32Kobayashi N. Boyer T.G. Berk A.J. Mol. Cell. Biol. 1995; 15: 6465-6473Google Scholar), TFIIH (33Xiao H. Pearson A. Coulombe B. Truant R. Zhang S. Regier J.L. Triezenberg S.J. Reinberg D. Flores O. Ingles C.J. Greenblatt J. Mol. Cell. Biol. 1994; 14: 7013-7024Google Scholar), and hTAFII32 (34Klemm R.D. Goodrich J.A. Zhou S. Tjian R. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 5788-5792Google Scholar). GAL4-VP16 was shown to stimulate three distinct events during the formation of the preinitiation complex: (i) formation of the TFIID-TFIIA-DNA complex (34Klemm R.D. Goodrich J.A. Zhou S. Tjian R. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 5788-5792Google Scholar); (ii) TFIIB recruitment (35Wang W. Gralla J.D. Carey M. Genes Dev. 1992; 6: 1716-1727Google Scholar); and, (iii) RAP30, TFIIEα, and RNAPII recruitment (36Choy B. Green M.R. Nature. 1993; 366: 531-536Google Scholar). A number of reports indicate that GAL4-VP16 can also act on open complex formation (37Wang W. Carey M. Gralla J.D. Science. 1992; 255: 450-453Google Scholar), transcript elongation (38Blau J. Xiao H. McCracken S. O'Hare P. Greenblatt J. Bentley D. Mol. Cell. Biol. 1996; 16: 2044-2055Google Scholar), and reinitiation (39Zawel L. Kumar K.P. Reinberg D. Genes Dev. 1995; 9: 1479-1490Google Scholar, 40Yudkovsky N. Ranish J.A. Hahn S. Nature. 2000; 408: 225-229Google Scholar). Using site-specific protein-DNA photo-cross-linking, we have analyzed the physical interaction between GAL4-VP16 molecules bound to promoter sites various distances from the TATA element and a complex containing TBP, TFIIB, and TFIIA assembled onto the TATA element. Using templates with either one or five GAL4 DNA-binding sites, we show that DNA-bound GAL4-VP16 does indeed cross-link to many positions in the vicinity of the TATA element where a TBP-TFIIA-TFIIB complex is assembled. Interaction with DNA-bound GAL4-VP16 does not significantly modify the conformation of the TBP-TFIIA-TFIIB-promoter complex. We also show that a GAL4-VP16 dimer bound to one GAL4 binding site can stimulate transcription with an efficiency approaching that of GAL4-VP16 dimers bound to five sites if the single dimer is properly positioned upstream of the TATA box. We thank members of our laboratory for helpful discussions, Diane Bourque for artwork, Vincent Trinh for the design of the molecular models in Fig. 6, and Will Home for critical reading of the manuscript.

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