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• W2007227478 abstract "Drosophila heat shock factor (HSF) binds to specific sequence elements of heat shock genes and can activate their transcription 200-fold. Though HSF has an acidic activation domain, the mechanistic details of heat shock gene activation remain undefined. Here we report that HSF interacts directly with the general transcription factor TBP (TATA-box binding protein), and these two factors bind cooperatively to heat shock promoters. A third factor that binds heat shock promoters, GAGA factor, also interacts with HSF and further stabilizes HSF binding to heat shock elements (HSEs). The interaction of HSF and TBP is explored in some detail here and is shown to be mediated by residues in both the amino- and carboxyl-terminal portions of HSF. This HSF/TBP interaction can be specifically disrupted by competition with the potent acidic transcriptional activator VP16. We further show that the acidic domain of the largest subunit of Drosophila RNA polymerase II (Pol II) associates with TBP in vitro and is specifically displaced from TBP upon addition of HSF. The region of TBP that mediates both HSF and Pol II acidic domain binding maps to the conserved carboxyl-terminal repeats and depends on at least one of the TBP residues known to be contacted by VP16 and to be critical for transcription activation. We discuss these findings in the context of a model in which HSF triggers hsp70 transcription by freeing the hsp70 promoter-paused Pol II from the constraints on elongation caused by the affinity of Pol II for general transcription factors. Drosophila heat shock factor (HSF) binds to specific sequence elements of heat shock genes and can activate their transcription 200-fold. Though HSF has an acidic activation domain, the mechanistic details of heat shock gene activation remain undefined. Here we report that HSF interacts directly with the general transcription factor TBP (TATA-box binding protein), and these two factors bind cooperatively to heat shock promoters. A third factor that binds heat shock promoters, GAGA factor, also interacts with HSF and further stabilizes HSF binding to heat shock elements (HSEs). The interaction of HSF and TBP is explored in some detail here and is shown to be mediated by residues in both the amino- and carboxyl-terminal portions of HSF. This HSF/TBP interaction can be specifically disrupted by competition with the potent acidic transcriptional activator VP16. We further show that the acidic domain of the largest subunit of Drosophila RNA polymerase II (Pol II) associates with TBP in vitro and is specifically displaced from TBP upon addition of HSF. The region of TBP that mediates both HSF and Pol II acidic domain binding maps to the conserved carboxyl-terminal repeats and depends on at least one of the TBP residues known to be contacted by VP16 and to be critical for transcription activation. We discuss these findings in the context of a model in which HSF triggers hsp70 transcription by freeing the hsp70 promoter-paused Pol II from the constraints on elongation caused by the affinity of Pol II for general transcription factors. Heat shock triggers formation of heat shock factor (HSF) 1The abbreviations used are: HSF, heat shock factor; Pol II, polymerase II; TBP, TATA-box binding protein; HSE, heat shock element; CTD, carboxyl-terminal domain; GST, glutathione S-transferase; DTT, dithiothreitol; GSH, glutathione; MBP, maltose-binding protein; BSA bovine serum albumin. 1The abbreviations used are: HSF, heat shock factor; Pol II, polymerase II; TBP, TATA-box binding protein; HSE, heat shock element; CTD, carboxyl-terminal domain; GST, glutathione S-transferase; DTT, dithiothreitol; GSH, glutathione; MBP, maltose-binding protein; BSA bovine serum albumin. protein trimers (1Westwood J.T. Clos J. Wu C. Nature. 1991; 353: 822-827Crossref PubMed Scopus (310) Google Scholar, 2Nakai A. Kawazoe Y. Tanabe M. Nagata K. Morimoto R.I. Mol. Cell. Biol. 1995; 15: 5268-5278Crossref PubMed Scopus (89) Google Scholar) that bind tightly to heat shock elements upstream of the hsp70 promoter. HSF binding is concomitant with a rapid and vigorous (200-fold) increase in the rate of transcription. The uninduced heat shock promoter is primed for rapid activation. This promoter is contained in a chromatin structure that is open and easily accessible and contains one paused Pol II per promoter (3Rasmussen E.B. Lis J.T. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 7923-7927Crossref PubMed Scopus (280) Google Scholar). A rate-limiting step in transcription appears to be the escape of this promoter-paused Pol II into productive elongation. Even after heat shock, when Pol II fires from the hsp70 promoter every 6 s, transient Pol II pausing can still be detected on hsp70 (4Giardina C. Perez R.M. Lis J.T. Genes Dev. 1992; 6: 2190-2200Crossref PubMed Scopus (131) Google Scholar, 5O'Brien T. Lis J.T. Mol. Cell. Biol. 1991; 11: 5285-5290Crossref PubMed Scopus (100) Google Scholar) such that Pol II progression through this specific region at the 5′ end of the transcription unit remains rate-limiting.The relative generality of transcriptional control at the level of paused polymerase (6Spencer C.A. Groudine M. Oncogene. 1990; 5: 777-785PubMed Google Scholar) indicates that many upstream activators can act to stimulate escape of the paused polymerase into a productive elongation mode. How might this happen? We favor a model in which the paused polymerase is restrained via its strong association with the promoter and general transcription factors. In this scenario, polymerase recruitment is vigorous while escape (beyond the pause) is limiting. The activator could act to increase the rate of Pol II escape by modifying the polymerase complex to produce an elongationally competent form or perhaps more simply by competing with Pol II for one or more binding sites on the general transcription apparatus. We have demonstrated previously that Pol II can bind TBP in vitroand can be displaced from TBP by competition with specific transcriptional activator proteins (VP16 and CTF1) (7Xiao H. Lis J.T. Nucleic Acids Res. 1994; 22: 1966-1973Crossref PubMed Scopus (55) Google Scholar, 8Xiao H. Lis J.T. Mol. Cell. Biol. 1994; 14: 7507-7516Crossref PubMed Scopus (36) Google Scholar).TBP is a good candidate for a heat shock factor target given its constitutive presence on hsp70 (4Giardina C. Perez R.M. Lis J.T. Genes Dev. 1992; 6: 2190-2200Crossref PubMed Scopus (131) Google Scholar) and given the close proximity of the TATA element to the proximal HSF binding sites. Also, the potent acidic activator VP16 has been shown to associate with TBP (9Ingles C.J. Shales M. Cress W.D. Triezenberg S.J. Greenblatt J. Nature. 1991; 351: 588-590Crossref PubMed Scopus (225) Google Scholar), and it is known that acidic activators like GAL4 can activate an hsp70 promoter in transgenic fly lines (10Fischer J.A. Giniger E. Maniatis T. Ptashne M. Nature. 1988; 332: 853-856Crossref PubMed Scopus (350) Google Scholar). Finally, the carboxyl-terminal domain (CTD) heptad repeats and the acidic domain (the so-called “H” domain) of RNA polymerase have been shown to interact genetically with each other (8Xiao H. Lis J.T. Mol. Cell. Biol. 1994; 14: 7507-7516Crossref PubMed Scopus (36) Google Scholar, 11Nonet M. Young R.A. Genetics. 1989; 123: 715-724Crossref PubMed Google Scholar), and both have been shown to bind to TBP in vitro (8Xiao H. Lis J.T. Mol. Cell. Biol. 1994; 14: 7507-7516Crossref PubMed Scopus (36) Google Scholar, 12Usheva A. Moldonado E. Goldring A. Lu H. Horbavi C. Reinberg D. Aloni Y. Cell. 1992; 69: 871-881Abstract Full Text PDF PubMed Scopus (179) Google Scholar). In our hands, the H-domain/TBP interaction is stronger than the CTD/TBP interaction.TBP-binding is only one of a variety of activities displayed by transcriptional activators. VP16 has been implicated in the recruitment of TFIIH (13Xiao 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: 7013-7024Crossref PubMed Scopus (327) Google Scholar). Since TFIIH has both DNA helicase activity and CTD-specific kinase activity, this suggests a role for activators in promoter melting and/or CTD phosphorylation. Such a modification of the polymerase complex might also play a role in the progression of the paused polymerase into productive elongation. VP16 has also been shown to associate with TFIIB in vitro (14Lin Y.S. Ha I. Moldonado E. Reinberg D. Green M.R. Nature. 1991; 353: 569-570Crossref PubMed Scopus (261) Google Scholar). The multiple interactions of activators with basal factors are consistent with multiple layers of activator-mediated regulation and the synergistic effect of activators (15Kakidani H. Ptashne M. Cell. 1988; 52: 161-167Abstract Full Text PDF PubMed Scopus (201) Google Scholar). A fraction of TBP is complexed in vivo, as TFIID, with at least eight TBP-associated factors (TAFs), which also have been implicated as promoter-specific activator targets (reviewed in Ref. 16Goodrich J.A. Tjian R. Curr. Opin. Cell Biol. 1994; 6: 403-409Crossref PubMed Scopus (210) Google Scholar). The TBP core of TFIID also serves as the foundation for assembly of the basal transcription apparatus (17Tang H. Sun X. Reinberg D. Ebright R. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 1119-1124Crossref PubMed Scopus (93) Google Scholar). TBP is, therefore, capable of many interactions, some of which must occur simultaneously. This may be possible if these interactions are specific for small portions of the TBP surface (as has been shown for several basal factor-TBP interactions, see Ref. 17Tang H. Sun X. Reinberg D. Ebright R. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 1119-1124Crossref PubMed Scopus (93) Google Scholar), allowing TBP to support additional activator contacts. Additionally, any of these protein-protein interactions may be quite dynamic, such that multiple factors could bind to the same site on the TBP surface.Here we present first tests of a simple “competition” hypothesis for heat shock gene regulation. Affinity chromatography assays demonstrate that HSF can bind to TBP in vitro and that this binding is competitive with the acidic H-domain of RNA polymerase. Portions of HSF and TBP that are required for this association are mapped. Competitive binding assays also suggest that HSF associates with TBP in a fashion similar to the potent acidic activator VP16. Additionally, it is shown that HSF and TBP bind cooperatively to heat shock promoters and that GAGA factor further stabilizes the HSF-HSE complex.DISCUSSIONWe have shown here that the heat shock gene-specific activator, HSF, binds efficiently to the general transcription factor TBP in vitro. In these experiments, comparable fractions of input TBP were recovered by HSF affinity chromatography using either Drosophila nuclear extracts or purified recombinant TBP. A second general factor TFIIB, which also has been reported to bind acidic activators (20Roberts G.E. Ha I. Maldonado E. Reinberg D. Green M.R. Nature. 1993; 363: 741-744Crossref PubMed Scopus (179) Google Scholar), shows only weak affinity for HSF. The HSF/TBP interaction appears to influence the association of these factors with their DNA targets, in that we observe that purified HSF and TBP bind cooperatively to heat shock promoters in vitro. Likewise, GAGA factor, another component of the hsp70 and hsp26 promoters, also aids the binding of HSF to the hsp70 promoter in vitro. Both TBP and GAGA factor occupy these heat shock promoters prior to induction by heat shock and are thus positioned to facilitate HSF recruitment. These interactions, coupled with the open chromatin configuration of heat shock promoters (33Wu C. Nature. 1980; 286: 854-860Crossref PubMed Scopus (747) Google Scholar), may help to explain the fact that HSF binding to HSEs in vivo is dependent on the presence of intact TFIID and GAGA binding sites (34Shopland L.S. Hirayoshi K. Fernandes M. Lis J.T. Genes Dev. 1995; 9: 2756-2769Crossref PubMed Scopus (112) Google Scholar). In addition, these interactions of HSF with TBP and GAGA factor may stabilize promoter associations of these factors during multiple rounds of activated transcription when proposed contacts of these factors with RNA polymerase II and other components of the basal machinery are likely to be disrupted during each cycle of transcription.The binding of HSF to TBP is mediated by residues in both the DNA-binding/trimerization domain and in the acidic carboxyl-terminal domain of HSF. This binding is targeted to the conserved carboxyl-terminal repeats of TBP. The binding of HSF to TBP is similar in both avidity and character to the binding of the acidic transcription activator VP16 to TBP. Both interactions are affected by a specific mutation in TBP (L114K). Moreover, VP16 and HSF compete for binding to TBP.We have also shown that an acidic domain (H) of DrosophilaRNA polymerase II binds to TBP in vitro in a manner similar to the polymerase/TBP interaction previously reported in yeast (8Xiao H. Lis J.T. Mol. Cell. Biol. 1994; 14: 7507-7516Crossref PubMed Scopus (36) Google Scholar). This interaction is disrupted upon addition of HSF, suggesting that polymerase and HSF can compete for the same site on TBP. This site on TBP also maps to the conserved TBP carboxyl-terminal repeats and is specifically reduced by the L114K mutation. We suggest that some of the same polymerase-general factor affinities that facilitate recruitment of Pol II to the promoter, like the TBP interaction seen here, also act as a “tether” that hinders polymerase escape into functional elongation and thus contribute to the formation of paused polymerase. These results provide the basis for a simple competition model for hsp70 transcriptional activation in which HSF frees the hsp70 paused polymerase from one component of the constraint on elongation caused by affinity of polymerase for general transcription factors at the core promoter.A model for activated transcription must, of course, account for multiple rounds of transcription. If HSF displaces a critical Pol II contact by binding to the core promoter complex, HSF may then occupy a site that is important for the next round of polymerase recruitment. How does the next Pol II molecule enter? Pol II recruitment appears to be accomplished via multiple known general factor contacts, including interactions involving TFIIF (35Flores O. Moldonado E. Reinberg D. J. Biol. Chem. 1989; 264: 8913-8921Abstract Full Text PDF PubMed Google Scholar), CTD with TBP (11Nonet M. Young R.A. Genetics. 1989; 123: 715-724Crossref PubMed Google Scholar), and polymerase with the promoter DNA itself. This recruitment rate would have to be much faster than Pol II escape to account for the observed full occupancy of the uninduced promoter by paused Pol II (4Giardina C. Perez R.M. Lis J.T. Genes Dev. 1992; 6: 2190-2200Crossref PubMed Scopus (131) Google Scholar). We propose that the TBP-binding activity of HSF increases the efficiency of the rate-limiting step (polymerase escape) while having a negligible inhibitory effect on the inherently fast polymerase recruitment provided by the multiple polymerase contacts of this strong promoter.While this competition model is attractive in its simplicity, it does not exclude other mechanisms that might act alternatively or additionally to increase the rate of escape of Pol II from the pause site into productive elongation. For example, HSF may facilitate recruitment of other general factors, which could modify the promoter-paused Pol II and thereby affect its escape to productive elongation. Furthermore, we have examined here only one of what may be several common contacts of HSF and Pol II with general factors.Eukaryotic transcription has many steps that can be fine tuned to the needs of the thousands of differentially regulated promoters. Many distinct regulatory steps have been documented, including TFIID-recruitment (36Xiao H. Friesen J.D. Lis J.T. Mol. Cell. Biol. 1995; 15: 5757-5761Crossref PubMed Scopus (102) Google Scholar, 37Chatterjee S. Struhl K. Nature. 1995; 374: 820-822Crossref PubMed Scopus (167) Google Scholar, 38Klages N. Strubin M. Nature. 1995; 374: 822-823Crossref PubMed Scopus (153) Google Scholar), Pol II recruitment (39Barberis A. Pearlberg J. Simkovich N. Farrell S. Reinagel P. Bamdad C. Sigal G. Ptashne M. Cell. 1995; 81: 359-368Abstract Full Text PDF PubMed Scopus (234) Google Scholar), promoter melting (40Pan G. Greenblatt J. J. Biol. Chem. 1994; 269: 30101-30104Abstract Full Text PDF PubMed Google Scholar), and elongational control after promoter escape (41Marshall N.F. Price D.H. Mol. Cell Biol. 1992; 12: 2078-2090Crossref PubMed Scopus (239) Google Scholar, 42Bradsher J.N. Jackson K.W. Conaway R.C. Conaway J.W. J. Biol. Chem. 1993; 268: 25587-25593Abstract Full Text PDF PubMed Google Scholar). In each case, the slow step in transcription must be the target of regulatory factors that either enhance or inhibit one of many specific molecular interactions required for establishing a productive transcription complex. The fact that regulatory factors and RNA polymerase interact with multiple general transcription factors provides the potential for modulation at any of multiple distinct steps. Heat shock triggers formation of heat shock factor (HSF) 1The abbreviations used are: HSF, heat shock factor; Pol II, polymerase II; TBP, TATA-box binding protein; HSE, heat shock element; CTD, carboxyl-terminal domain; GST, glutathione S-transferase; DTT, dithiothreitol; GSH, glutathione; MBP, maltose-binding protein; BSA bovine serum albumin. 1The abbreviations used are: HSF, heat shock factor; Pol II, polymerase II; TBP, TATA-box binding protein; HSE, heat shock element; CTD, carboxyl-terminal domain; GST, glutathione S-transferase; DTT, dithiothreitol; GSH, glutathione; MBP, maltose-binding protein; BSA bovine serum albumin. protein trimers (1Westwood J.T. Clos J. Wu C. Nature. 1991; 353: 822-827Crossref PubMed Scopus (310) Google Scholar, 2Nakai A. Kawazoe Y. Tanabe M. Nagata K. Morimoto R.I. Mol. Cell. Biol. 1995; 15: 5268-5278Crossref PubMed Scopus (89) Google Scholar) that bind tightly to heat shock elements upstream of the hsp70 promoter. HSF binding is concomitant with a rapid and vigorous (200-fold) increase in the rate of transcription. The uninduced heat shock promoter is primed for rapid activation. This promoter is contained in a chromatin structure that is open and easily accessible and contains one paused Pol II per promoter (3Rasmussen E.B. Lis J.T. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 7923-7927Crossref PubMed Scopus (280) Google Scholar). A rate-limiting step in transcription appears to be the escape of this promoter-paused Pol II into productive elongation. Even after heat shock, when Pol II fires from the hsp70 promoter every 6 s, transient Pol II pausing can still be detected on hsp70 (4Giardina C. Perez R.M. Lis J.T. Genes Dev. 1992; 6: 2190-2200Crossref PubMed Scopus (131) Google Scholar, 5O'Brien T. Lis J.T. Mol. Cell. Biol. 1991; 11: 5285-5290Crossref PubMed Scopus (100) Google Scholar) such that Pol II progression through this specific region at the 5′ end of the transcription unit remains rate-limiting. The relative generality of transcriptional control at the level of paused polymerase (6Spencer C.A. Groudine M. Oncogene. 1990; 5: 777-785PubMed Google Scholar) indicates that many upstream activators can act to stimulate escape of the paused polymerase into a productive elongation mode. How might this happen? We favor a model in which the paused polymerase is restrained via its strong association with the promoter and general transcription factors. In this scenario, polymerase recruitment is vigorous while escape (beyond the pause) is limiting. The activator could act to increase the rate of Pol II escape by modifying the polymerase complex to produce an elongationally competent form or perhaps more simply by competing with Pol II for one or more binding sites on the general transcription apparatus. We have demonstrated previously that Pol II can bind TBP in vitroand can be displaced from TBP by competition with specific transcriptional activator proteins (VP16 and CTF1) (7Xiao H. Lis J.T. Nucleic Acids Res. 1994; 22: 1966-1973Crossref PubMed Scopus (55) Google Scholar, 8Xiao H. Lis J.T. Mol. Cell. Biol. 1994; 14: 7507-7516Crossref PubMed Scopus (36) Google Scholar). TBP is a good candidate for a heat shock factor target given its constitutive presence on hsp70 (4Giardina C. Perez R.M. Lis J.T. Genes Dev. 1992; 6: 2190-2200Crossref PubMed Scopus (131) Google Scholar) and given the close proximity of the TATA element to the proximal HSF binding sites. Also, the potent acidic activator VP16 has been shown to associate with TBP (9Ingles C.J. Shales M. Cress W.D. Triezenberg S.J. Greenblatt J. Nature. 1991; 351: 588-590Crossref PubMed Scopus (225) Google Scholar), and it is known that acidic activators like GAL4 can activate an hsp70 promoter in transgenic fly lines (10Fischer J.A. Giniger E. Maniatis T. Ptashne M. Nature. 1988; 332: 853-856Crossref PubMed Scopus (350) Google Scholar). Finally, the carboxyl-terminal domain (CTD) heptad repeats and the acidic domain (the so-called “H” domain) of RNA polymerase have been shown to interact genetically with each other (8Xiao H. Lis J.T. Mol. Cell. Biol. 1994; 14: 7507-7516Crossref PubMed Scopus (36) Google Scholar, 11Nonet M. Young R.A. Genetics. 1989; 123: 715-724Crossref PubMed Google Scholar), and both have been shown to bind to TBP in vitro (8Xiao H. Lis J.T. Mol. Cell. Biol. 1994; 14: 7507-7516Crossref PubMed Scopus (36) Google Scholar, 12Usheva A. Moldonado E. Goldring A. Lu H. Horbavi C. Reinberg D. Aloni Y. Cell. 1992; 69: 871-881Abstract Full Text PDF PubMed Scopus (179) Google Scholar). In our hands, the H-domain/TBP interaction is stronger than the CTD/TBP interaction. TBP-binding is only one of a variety of activities displayed by transcriptional activators. VP16 has been implicated in the recruitment of TFIIH (13Xiao 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: 7013-7024Crossref PubMed Scopus (327) Google Scholar). Since TFIIH has both DNA helicase activity and CTD-specific kinase activity, this suggests a role for activators in promoter melting and/or CTD phosphorylation. Such a modification of the polymerase complex might also play a role in the progression of the paused polymerase into productive elongation. VP16 has also been shown to associate with TFIIB in vitro (14Lin Y.S. Ha I. Moldonado E. Reinberg D. Green M.R. Nature. 1991; 353: 569-570Crossref PubMed Scopus (261) Google Scholar). The multiple interactions of activators with basal factors are consistent with multiple layers of activator-mediated regulation and the synergistic effect of activators (15Kakidani H. Ptashne M. Cell. 1988; 52: 161-167Abstract Full Text PDF PubMed Scopus (201) Google Scholar). A fraction of TBP is complexed in vivo, as TFIID, with at least eight TBP-associated factors (TAFs), which also have been implicated as promoter-specific activator targets (reviewed in Ref. 16Goodrich J.A. Tjian R. Curr. Opin. Cell Biol. 1994; 6: 403-409Crossref PubMed Scopus (210) Google Scholar). The TBP core of TFIID also serves as the foundation for assembly of the basal transcription apparatus (17Tang H. Sun X. Reinberg D. Ebright R. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 1119-1124Crossref PubMed Scopus (93) Google Scholar). TBP is, therefore, capable of many interactions, some of which must occur simultaneously. This may be possible if these interactions are specific for small portions of the TBP surface (as has been shown for several basal factor-TBP interactions, see Ref. 17Tang H. Sun X. Reinberg D. Ebright R. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 1119-1124Crossref PubMed Scopus (93) Google Scholar), allowing TBP to support additional activator contacts. Additionally, any of these protein-protein interactions may be quite dynamic, such that multiple factors could bind to the same site on the TBP surface. Here we present first tests of a simple “competition” hypothesis for heat shock gene regulation. Affinity chromatography assays demonstrate that HSF can bind to TBP in vitro and that this binding is competitive with the acidic H-domain of RNA polymerase. Portions of HSF and TBP that are required for this association are mapped. Competitive binding assays also suggest that HSF associates with TBP in a fashion similar to the potent acidic activator VP16. Additionally, it is shown that HSF and TBP bind cooperatively to heat shock promoters and that GAGA factor further stabilizes the HSF-HSE complex. DISCUSSIONWe have shown here that the heat shock gene-specific activator, HSF, binds efficiently to the general transcription factor TBP in vitro. In these experiments, comparable fractions of input TBP were recovered by HSF affinity chromatography using either Drosophila nuclear extracts or purified recombinant TBP. A second general factor TFIIB, which also has been reported to bind acidic activators (20Roberts G.E. Ha I. Maldonado E. Reinberg D. Green M.R. Nature. 1993; 363: 741-744Crossref PubMed Scopus (179) Google Scholar), shows only weak affinity for HSF. The HSF/TBP interaction appears to influence the association of these factors with their DNA targets, in that we observe that purified HSF and TBP bind cooperatively to heat shock promoters in vitro. Likewise, GAGA factor, another component of the hsp70 and hsp26 promoters, also aids the binding of HSF to the hsp70 promoter in vitro. Both TBP and GAGA factor occupy these heat shock promoters prior to induction by heat shock and are thus positioned to facilitate HSF recruitment. These interactions, coupled with the open chromatin configuration of heat shock promoters (33Wu C. Nature. 1980; 286: 854-860Crossref PubMed Scopus (747) Google Scholar), may help to explain the fact that HSF binding to HSEs in vivo is dependent on the presence of intact TFIID and GAGA binding sites (34Shopland L.S. Hirayoshi K. Fernandes M. Lis J.T. Genes Dev. 1995; 9: 2756-2769Crossref PubMed Scopus (112) Google Scholar). In addition, these interactions of HSF with TBP and GAGA factor may stabilize promoter associations of these factors during multiple rounds of activated transcription when proposed contacts of these factors with RNA polymerase II and other components of the basal machinery are likely to be disrupted during each cycle of transcription.The binding of HSF to TBP is mediated by residues in both the DNA-binding/trimerization domain and in the acidic carboxyl-terminal domain of HSF. This binding is targeted to the conserved carboxyl-terminal repeats of TBP. The binding of HSF to TBP is similar in both avidity and character to the binding of the acidic transcription activator VP16 to TBP. Both interactions are affected by a specific mutation in TBP (L114K). Moreover, VP16 and HSF compete for binding to TBP.We have also shown that an acidic domain (H) of DrosophilaRNA polymerase II binds to TBP in vitro in a manner similar to the polymerase/TBP interaction previously reported in yeast (8Xiao H. Lis J.T. Mol. Cell. Biol. 1994; 14: 7507-7516Crossref PubMed Scopus (36) Google Scholar). This interaction is disrupted upon addition of HSF, suggesting that polymerase and HSF can compete for the same site on TBP. This site on TBP also maps to the conserved TBP carboxyl-terminal repeats and is specifically reduced by the L114K mutation. We suggest that some of the same polymerase-general factor affinities that facilitate recruitment of Pol II to the promoter, like the TBP interaction seen here, also act as a “tether” that hinders polymerase escape into functional elongation and thus contribute to the formation of paused polymerase. These results provide the basis for a simple competition model for hsp70 transcriptional activation in which HSF frees the hsp70 paused polymerase from one component of the constraint on elongation caused by affinity of polymerase for general transcription factors at the core promoter.A model for activated transcription must, of course, account for multiple rounds of transcription. If HSF displaces a critical Pol II contact by binding to the core promoter complex, HSF may then occupy a site that is important for the next round of polymerase recruitment. How does the next Pol II molecule enter? Pol II recruitment appears to be accomplished via multiple known general factor contacts, including interactions involving TFIIF (35Flores O. Moldonado E. Reinberg D. J. Biol. Chem. 1989; 264: 8913-8921Abstract Full Text PDF PubMed Google Scholar), CTD with TBP (11Nonet M. Young R.A. Genetics. 1989; 123: 715-724Crossref PubMed Google Scholar), and polymerase with the promoter DNA itself. This recruitment rate would have to be much faster than Pol II escape to account for the observed full occupancy of the uninduced promoter by paused Pol II (4Giardina C. Perez R.M. Lis J.T. Genes Dev. 1992; 6: 2190-2200Crossref PubMed Scopus (131) Google Scholar). We propose that the TBP-binding activity of HSF increases the efficiency of the rate-limiting step (polymerase escape) while having a negligible inhibitory effect on the inherently fast polymerase recruitment provided by the multiple polymerase contacts of this strong promoter.While this competition model is attractive in its simplicity, it does not exclude other mechanisms that might act alternatively or additionally to increase the rate of escape of Pol II from the pause site into productive elongation. For example, HSF may facilitate recruitment of other general factors, which could modify the promoter-paused Pol II and thereby affect its escape to productive elongation. Furthermore, we have examined here only one of what may be several common contacts of HSF and Pol II with general factors.Eukaryotic transcription has many steps that can be fine tuned to the needs of the thousands of differentially regulated promoters. Many distinct regulatory steps have been documented, including TFIID-recruitment (36Xiao H. Friesen J.D. Lis J.T. Mol. Cell. Biol. 1995; 15: 5757-5761Crossref PubMed Scopus (102) Google Scholar, 37Chatterjee S. Struhl K. Nature. 1995; 374: 820-822Crossref PubMed Scopus (167) Google Scholar, 38Klages N. Strubin M. Nature. 1995; 374: 822-823Crossref PubMed Scopus (153) Google Scholar), Pol II recruitment (39Barberis A. Pearlberg J. Simkovich N. Farrell S. Reinagel P. Bamdad C. Sigal G. Ptashne M. Cell. 1995; 81: 359-368Abstract Full Text PDF PubMed Scopus (234) Google Scholar), promoter melting (40Pan G. Greenblatt J. J. Biol. Chem. 1994; 269: 30101-30104Abstract Full Text PDF PubMed Google Scholar), and elongational control after promoter escape (41Marshall N.F. Price D.H. Mol. Cell Biol. 1992; 12: 2078-2090Crossref PubMed Scopus (239) Google Scholar, 42Bradsher J.N. Jackson K.W. Conaway R.C. Conaway J.W. J. Biol. Chem. 1993; 268: 25587-25593Abstract Full Text PDF PubMed Google Scholar). In each case, the slow step in transcription must be the target of regulatory factors that either enhance or inhibit one of many specific molecular interactions required for establishing a productive transcription complex. The fact that regulatory factors and RNA polymerase interact with multiple general transcription factors provides the potential for modulation at any of multiple distinct steps. We have shown here that the heat shock gene-specific activator, HSF, binds efficiently to the general transcription factor TBP in vitro. In these experiments, comparable fractions of input TBP were recovered by HSF affinity chromatography using either Drosophila nuclear extracts or purified recombinant TBP. A second general factor TFIIB, which also has been reported to bind acidic activators (20Roberts G.E. Ha I. Maldonado E. Reinberg D. Green M.R. Nature. 1993; 363: 741-744Crossref PubMed Scopus (179) Google Scholar), shows only weak affinity for HSF. The HSF/TBP interaction appears to influence the association of these factors with their DNA targets, in that we observe that purified HSF and TBP bind cooperatively to heat shock promoters in vitro. Likewise, GAGA factor, another component of the hsp70 and hsp26 promoters, also aids the binding of HSF to the hsp70 promoter in vitro. Both TBP and GAGA factor occupy these heat shock promoters prior to induction by heat shock and are thus positioned to facilitate HSF recruitment. These interactions, coupled with the open chromatin configuration of heat shock promoters (33Wu C. Nature. 1980; 286: 854-860Crossref PubMed Scopus (747) Google Scholar), may help to explain the fact that HSF binding to HSEs in vivo is dependent on the presence of intact TFIID and GAGA binding sites (34Shopland L.S. Hirayoshi K. Fernandes M. Lis J.T. Genes Dev. 1995; 9: 2756-2769Crossref PubMed Scopus (112) Google Scholar). In addition, these interactions of HSF with TBP and GAGA factor may stabilize promoter associations of these factors during multiple rounds of activated transcription when proposed contacts of these factors with RNA polymerase II and other components of the basal machinery are likely to be disrupted during each cycle of transcription. The binding of HSF to TBP is mediated by residues in both the DNA-binding/trimerization domain and in the acidic carboxyl-terminal domain of HSF. This binding is targeted to the conserved carboxyl-terminal repeats of TBP. The binding of HSF to TBP is similar in both avidity and character to the binding of the acidic transcription activator VP16 to TBP. Both interactions are affected by a specific mutation in TBP (L114K). Moreover, VP16 and HSF compete for binding to TBP. We have also shown that an acidic domain (H) of DrosophilaRNA polymerase II binds to TBP in vitro in a manner similar to the polymerase/TBP interaction previously reported in yeast (8Xiao H. Lis J.T. Mol. Cell. Biol. 1994; 14: 7507-7516Crossref PubMed Scopus (36) Google Scholar). This interaction is disrupted upon addition of HSF, suggesting that polymerase and HSF can compete for the same site on TBP. This site on TBP also maps to the conserved TBP carboxyl-terminal repeats and is specifically reduced by the L114K mutation. We suggest that some of the same polymerase-general factor affinities that facilitate recruitment of Pol II to the promoter, like the TBP interaction seen here, also act as a “tether” that hinders polymerase escape into functional elongation and thus contribute to the formation of paused polymerase. These results provide the basis for a simple competition model for hsp70 transcriptional activation in which HSF frees the hsp70 paused polymerase from one component of the constraint on elongation caused by affinity of polymerase for general transcription factors at the core promoter. A model for activated transcription must, of course, account for multiple rounds of transcription. If HSF displaces a critical Pol II contact by binding to the core promoter complex, HSF may then occupy a site that is important for the next round of polymerase recruitment. How does the next Pol II molecule enter? Pol II recruitment appears to be accomplished via multiple known general factor contacts, including interactions involving TFIIF (35Flores O. Moldonado E. Reinberg D. J. Biol. Chem. 1989; 264: 8913-8921Abstract Full Text PDF PubMed Google Scholar), CTD with TBP (11Nonet M. Young R.A. Genetics. 1989; 123: 715-724Crossref PubMed Google Scholar), and polymerase with the promoter DNA itself. This recruitment rate would have to be much faster than Pol II escape to account for the observed full occupancy of the uninduced promoter by paused Pol II (4Giardina C. Perez R.M. Lis J.T. Genes Dev. 1992; 6: 2190-2200Crossref PubMed Scopus (131) Google Scholar). We propose that the TBP-binding activity of HSF increases the efficiency of the rate-limiting step (polymerase escape) while having a negligible inhibitory effect on the inherently fast polymerase recruitment provided by the multiple polymerase contacts of this strong promoter. While this competition model is attractive in its simplicity, it does not exclude other mechanisms that might act alternatively or additionally to increase the rate of escape of Pol II from the pause site into productive elongation. For example, HSF may facilitate recruitment of other general factors, which could modify the promoter-paused Pol II and thereby affect its escape to productive elongation. Furthermore, we have examined here only one of what may be several common contacts of HSF and Pol II with general factors. Eukaryotic transcription has many steps that can be fine tuned to the needs of the thousands of differentially regulated promoters. Many distinct regulatory steps have been documented, including TFIID-recruitment (36Xiao H. Friesen J.D. Lis J.T. Mol. Cell. Biol. 1995; 15: 5757-5761Crossref PubMed Scopus (102) Google Scholar, 37Chatterjee S. Struhl K. Nature. 1995; 374: 820-822Crossref PubMed Scopus (167) Google Scholar, 38Klages N. Strubin M. Nature. 1995; 374: 822-823Crossref PubMed Scopus (153) Google Scholar), Pol II recruitment (39Barberis A. Pearlberg J. Simkovich N. Farrell S. Reinagel P. Bamdad C. Sigal G. Ptashne M. Cell. 1995; 81: 359-368Abstract Full Text PDF PubMed Scopus (234) Google Scholar), promoter melting (40Pan G. Greenblatt J. J. Biol. Chem. 1994; 269: 30101-30104Abstract Full Text PDF PubMed Google Scholar), and elongational control after promoter escape (41Marshall N.F. Price D.H. Mol. Cell Biol. 1992; 12: 2078-2090Crossref PubMed Scopus (239) Google Scholar, 42Bradsher J.N. Jackson K.W. Conaway R.C. Conaway J.W. J. Biol. Chem. 1993; 268: 25587-25593Abstract Full Text PDF PubMed Google Scholar). In each case, the slow step in transcription must be the target of regulatory factors that either enhance or inhibit one of many specific molecular interactions required for establishing a productive transcription complex. The fact that regulatory factors and RNA polymerase interact with multiple general transcription factors provides the potential for modulation at any of multiple distinct steps. We thank C. Wu, A. Greenleaf, R. Roeder, K. Struhl, and R. Tjian for plasmids, S. Buratowski for anti-yTBP antiserum, J. Kadonaga for anti-dTFIIB antiserum, R. C. Wilkins for anti-GAGA antiserum, and T. O'Brien and R. Tjian for purified recombinant dTFIIB and dTFIIA. We also thank V. Vogt, J. Helmann, J. Lin, and H. Shi for critical reading of this manuscript and members of the Lis laboratory for helpful discussions." @default.
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