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• W2002899891 abstract "Assembly of transcription pre-initiation complexes proceeds from the initial complex formed between “TATA” bearing promoter DNA and the TATA-binding protein (TBP). Our laboratory has been investigating the relationships among TATA sequence, TBP·TATA solution structure, recognition mechanisms, and transcription efficiency. TBP·TATA interactions have been modeled by global analysis of detailed kinetic and thermodynamic data obtained using fluorimetric and fluorometric techniques in conjunction with fluorescence resonance energy transfer. We have reported recently that TBP recognition of two consensus promoters, adenovirus major late (AdMLP: TATAAAAG) and E4 (TATATATA), is well described by a linear two-intermediate mechanism with simultaneous DNA binding and bending. Similar DNA geometries and high transcription efficiencies characterize these TBP·TATA complexes. Here we show that, in contrast to the consensus sequences, TBP recognition of a variant sequence (C7: TATAAACG) is described by a three-step model with two branching pathways. One pathway proceeds through an intermediate having severely bent DNA, reminiscent of the consensus interactions, with the other branch yielding a unique conformer with shallowly bent DNA. The resulting TBP·C7 complex has a dramatically different solution conformation than for TBP·DNACONSENSUS and is correlated with diminished relative transcription activity. The temperature dependence of the TBP·C7 helical bend is postulated to derive from population shifts between the conformers with slightly and severely bent DNA. Assembly of transcription pre-initiation complexes proceeds from the initial complex formed between “TATA” bearing promoter DNA and the TATA-binding protein (TBP). Our laboratory has been investigating the relationships among TATA sequence, TBP·TATA solution structure, recognition mechanisms, and transcription efficiency. TBP·TATA interactions have been modeled by global analysis of detailed kinetic and thermodynamic data obtained using fluorimetric and fluorometric techniques in conjunction with fluorescence resonance energy transfer. We have reported recently that TBP recognition of two consensus promoters, adenovirus major late (AdMLP: TATAAAAG) and E4 (TATATATA), is well described by a linear two-intermediate mechanism with simultaneous DNA binding and bending. Similar DNA geometries and high transcription efficiencies characterize these TBP·TATA complexes. Here we show that, in contrast to the consensus sequences, TBP recognition of a variant sequence (C7: TATAAACG) is described by a three-step model with two branching pathways. One pathway proceeds through an intermediate having severely bent DNA, reminiscent of the consensus interactions, with the other branch yielding a unique conformer with shallowly bent DNA. The resulting TBP·C7 complex has a dramatically different solution conformation than for TBP·DNACONSENSUS and is correlated with diminished relative transcription activity. The temperature dependence of the TBP·C7 helical bend is postulated to derive from population shifts between the conformers with slightly and severely bent DNA. TATA-binding protein fluorescence resonance energy transfer carboxytetramethylrhodamine fluorescein double-labeled duplex containing the C7 sequence (TAMRA-5′GGGCTATAAACGGG3′-fluorescein) single-labeled duplex containing the C7 sequence unlabeled duplex GGGCTATATATAGG GGGCTATAAAAGGG binary complex formed between TBP and DNA For eukaryotic class II genes, the binary complex formed between the TATA-binding protein and a conserved promoter site, the TATA box, provides the foundation upon which the transcription pre-initiation complex is assembled (1Burley S.K. Roeder R.G. Annu. Rev. Biochem. 1996; 65: 769-799Crossref PubMed Scopus (620) Google Scholar, 2Hernandez N. Genes Dev. 1993; 7: 1291-1308Crossref PubMed Scopus (560) Google Scholar, 3Horikoshi M. Bertuccioli C. Takada R. Wang J. Yamamoto T. Roeder R.G. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 1060-1064Crossref PubMed Scopus (110) Google Scholar, 4Maldonado E. Reinberg D. Curr. Opin. Cell Biol. 1995; 7: 352-361Crossref PubMed Scopus (78) Google Scholar, 5McKnight S.L. Genes Dev. 1996; 10: 367-381Crossref PubMed Scopus (49) Google Scholar, 6Oelgeschlager T. Chiang C.M. Roeder R.G. Nature. 1996; 382: 735-738Crossref PubMed Scopus (149) Google Scholar, 7Tang H. Sun X. Reinberg D. Ebright R.H. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 1119-1124Crossref PubMed Scopus (93) Google Scholar). TBP1 binds productively to consensus (TATA(a/t)A(a/t)N) and diverse variant TATA sequences (8Hahn S. Buratowski S. Sharp P.A. Guarente L. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 5718-5722Crossref PubMed Scopus (213) Google Scholar, 9Wobbe C.R. Struhl K. Mol. Cell. Biol. 1990; 10: 3859-3867Crossref PubMed Scopus (238) Google Scholar), yielding relative transcription efficiencies ranging from <1 to 172 (9Wobbe C.R. Struhl K. Mol. Cell. Biol. 1990; 10: 3859-3867Crossref PubMed Scopus (238) Google Scholar, 10Starr D.B. Hoopes B.C. Hawley D.K. J. Mol. Biol. 1995; 250: 434-446Crossref PubMed Scopus (158) Google Scholar). The interactions of Saccharomyces cerevisiae TBP with promoters bearing two consensus sequences, adenovirus major late (TATAAAAG, AdMLP) and E4 (TATATATA), have been well characterized via extensive biochemical (11Cox J.M. Hayward M.M. Sanchez J.F. Gegnas L.D. van der Zee S. Dennis J.H. Sigler P.B. Schepartz A. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 13475-13480Crossref PubMed Scopus (63) Google Scholar, 12Hoopes B.C. LeBlanc J.F. Hawley D.K. J. Biol. Chem. 1992; 267: 11539-11547Abstract Full Text PDF PubMed Google Scholar, 13Coleman R.A. Pugh B.F. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 7221-7226Crossref PubMed Scopus (59) Google Scholar, 14Liu Y. Schepartz A. Biochemistry. 2001; 40: 6257-6266Crossref PubMed Scopus (11) Google Scholar, 15Parkhurst K.M. Brenowitz M. Parkhurst L.J. Biochemistry. 1996; 35: 7459-7465Crossref PubMed Scopus (119) Google Scholar, 16Parkhurst K.M. Richards R.M. Brenowitz M. Parkhurst L.J. J. Mol. Biol. 1999; 289: 1327-1341Crossref PubMed Scopus (72) Google Scholar, 17Perez-Howard G.M. Weil P.A. Beechem J.M. Biochemistry. 1995; 34: 8005-8017Crossref PubMed Scopus (131) Google Scholar, 18Petri V. Hsieh M. Brenowitz M. Biochemistry. 1995; 34: 9977-9984Crossref PubMed Scopus (89) Google Scholar, 19Petri V. Hsieh M. Jamison E. Brenowitz M. Biochemistry. 1998; 37: 15842-15849Crossref PubMed Scopus (45) Google Scholar, 20Powell R.M. Parkhurst K.M. Brenowitz M. Parkhurst L.J. J. Biol. Chem. 2001; 276: 29782-29791Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar, 21Wolner B.S. Gralla J.D. J. Biol. Chem. 2001; 276: 6260-6266Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar, 22Wolner B.S. Gralla J.D. Mol. Cell. Biol. 2000; 20: 3608-3615Crossref PubMed Scopus (21) Google Scholar), crystallographic (23Juo Z.S. Chiu T.K. Leiberman P.M. Baikalov I. Berk A.J. Dickerson R.E. J. Mol. Biol. 1996; 261: 239-254Crossref PubMed Scopus (281) Google Scholar, 24Kim J.L. Nikolov D.B. Burley S.K. Nature. 1993; 365: 520-527Crossref PubMed Scopus (962) Google Scholar, 25Kim Y. Geiger J.H. Hahn S. Sigler P.B. Nature. 1993; 365: 512-520Crossref PubMed Scopus (1005) Google Scholar, 26Kim J.L. Burley S.K. Nat. Struct. Biol. 1994; 1: 638-653Crossref PubMed Scopus (197) Google Scholar, 27Nikolov D.B. Chen H. Halay E.D. Hoffman A. Roeder R.G. Burley S.K. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 4862-4867Crossref PubMed Scopus (255) Google Scholar, 28Patikoglou G.A. Kim J.L. Sun L. Yang S.H. Kodadek T. Burley S.K. Genes Dev. 1999; 13: 3217-3230Crossref PubMed Scopus (233) Google Scholar), and molecular dynamics (29de Souza O.N. Ornstein R.L. Biopolymers. 1998; 46: 403-415Crossref PubMed Scopus (37) Google Scholar, 30Pardo L. Pastor N. Weinstein H. Biophys. J. 1998; 75: 2411-2421Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar, 31Pastor N. Weinstein H. Jamison E. Brenowitz M. J. Mol. Biol. 2000; 304: 55-68Crossref PubMed Scopus (131) Google Scholar, 32Pastor N. Pardo L. Weinstein H. Biophys. J. 1997; 73: 640-652Abstract Full Text PDF PubMed Scopus (37) Google Scholar, 33Qian X. Strahs D. Schlick T. J. Mol. Biol. 2001; 308: 681-703Crossref PubMed Scopus (26) Google Scholar) studies. Both promoters bind to TBP at rates significantly slower than diffusion limited (12Hoopes B.C. LeBlanc J.F. Hawley D.K. J. Biol. Chem. 1992; 267: 11539-11547Abstract Full Text PDF PubMed Google Scholar, 13Coleman R.A. Pugh B.F. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 7221-7226Crossref PubMed Scopus (59) Google Scholar, 15Parkhurst K.M. Brenowitz M. Parkhurst L.J. Biochemistry. 1996; 35: 7459-7465Crossref PubMed Scopus (119) Google Scholar, 16Parkhurst K.M. Richards R.M. Brenowitz M. Parkhurst L.J. J. Mol. Biol. 1999; 289: 1327-1341Crossref PubMed Scopus (72) Google Scholar, 17Perez-Howard G.M. Weil P.A. Beechem J.M. Biochemistry. 1995; 34: 8005-8017Crossref PubMed Scopus (131) Google Scholar, 18Petri V. Hsieh M. Brenowitz M. Biochemistry. 1995; 34: 9977-9984Crossref PubMed Scopus (89) Google Scholar, 19Petri V. Hsieh M. Jamison E. Brenowitz M. Biochemistry. 1998; 37: 15842-15849Crossref PubMed Scopus (45) Google Scholar, 20Powell R.M. Parkhurst K.M. Brenowitz M. Parkhurst L.J. J. Biol. Chem. 2001; 276: 29782-29791Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar), forming tightly bound complexes with similar co-crystal structures (23Juo Z.S. Chiu T.K. Leiberman P.M. Baikalov I. Berk A.J. Dickerson R.E. J. Mol. Biol. 1996; 261: 239-254Crossref PubMed Scopus (281) Google Scholar, 24Kim J.L. Nikolov D.B. Burley S.K. Nature. 1993; 365: 520-527Crossref PubMed Scopus (962) Google Scholar, 25Kim Y. Geiger J.H. Hahn S. Sigler P.B. Nature. 1993; 365: 512-520Crossref PubMed Scopus (1005) Google Scholar, 26Kim J.L. Burley S.K. Nat. Struct. Biol. 1994; 1: 638-653Crossref PubMed Scopus (197) Google Scholar, 27Nikolov D.B. Chen H. Halay E.D. Hoffman A. Roeder R.G. Burley S.K. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 4862-4867Crossref PubMed Scopus (255) Google Scholar) and comparable DNA geometries in solution (34Wu J. Parkhurst K.M. Powell R.M. Brenowitz M. Parkhurst L.J. J. Biol. Chem. 2001; 276: 14614-14622Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). Additionally, these two sequences yield high relative transcription activities (9Wobbe C.R. Struhl K. Mol. Cell. Biol. 1990; 10: 3859-3867Crossref PubMed Scopus (238) Google Scholar,10Starr D.B. Hoopes B.C. Hawley D.K. J. Mol. Biol. 1995; 250: 434-446Crossref PubMed Scopus (158) Google Scholar). Our laboratory has been studying the detailed recognition mechanism of DNA promoters by S. cerevisiae TBP (15Parkhurst K.M. Brenowitz M. Parkhurst L.J. Biochemistry. 1996; 35: 7459-7465Crossref PubMed Scopus (119) Google Scholar, 16Parkhurst K.M. Richards R.M. Brenowitz M. Parkhurst L.J. J. Mol. Biol. 1999; 289: 1327-1341Crossref PubMed Scopus (72) Google Scholar, 20Powell R.M. Parkhurst K.M. Brenowitz M. Parkhurst L.J. J. Biol. Chem. 2001; 276: 29782-29791Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar) and the solution geometries of the resulting TBP·TATA complexes (34Wu J. Parkhurst K.M. Powell R.M. Brenowitz M. Parkhurst L.J. J. Biol. Chem. 2001; 276: 14614-14622Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 35Wu J. Parkhurst K.M. Powell R.M. Parkhurst L.J. J. Biol. Chem. 2001; 276: 14623-14627Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). Both lines of investigation utilize dye-labeled TATA-bearing oligomers and steady-state, stopped-flow, and time-resolved fluorescence techniques in conjunction with fluorescence resonance energy transfer (FRET). Global analysis of extensive real-time kinetic and thermodynamic data sets first revealed a linear three-step mechanism for the TBP·AdMLP reaction (16Parkhurst K.M. Richards R.M. Brenowitz M. Parkhurst L.J. J. Mol. Biol. 1999; 289: 1327-1341Crossref PubMed Scopus (72) Google Scholar), with intermediate conformers having DNA bent to the same extent as in the final complex. These conformers are present at high mole fraction throughout the reaction and persist at equilibrium. The parallel investigation of TBP·DNA solution structures concurrently revealed a strong sequence dependence of the DNA helical bend in such complexes (34Wu J. Parkhurst K.M. Powell R.M. Brenowitz M. Parkhurst L.J. J. Biol. Chem. 2001; 276: 14614-14622Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). Because the latter suggested TATA sequence-dependent recognition interactions, the mechanistic study was extended beyond AdMLP to include other sequences. We first asked the question, “How do the detailed recognition processes of different consensus sequences by TBP compare?” The E4 sequence was chosen as the alternate consensus sequence, because functional differences between the TBP·AdMLP and TBP·E4 interactions had been identified previously using DNase I footprinting (18Petri V. Hsieh M. Brenowitz M. Biochemistry. 1995; 34: 9977-9984Crossref PubMed Scopus (89) Google Scholar, 19Petri V. Hsieh M. Jamison E. Brenowitz M. Biochemistry. 1998; 37: 15842-15849Crossref PubMed Scopus (45) Google Scholar). Extensive data collection and model fitting using global analysis showed the linear two-intermediate model to be common to the reaction of TBP with both strong promoters (20Powell R.M. Parkhurst K.M. Brenowitz M. Parkhurst L.J. J. Biol. Chem. 2001; 276: 29782-29791Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). The energetics of the partial reactions for the two promoters was very similar for the initial step, formation of the first intermediate conformer. Beyond that initial binding and bending event, however, the reaction progression differed substantially, with the TBP·E4 interaction nearly complete after the second step but with the TBP·AdMLP reaction continuing to undergo large energetic changes in the final step. Having thus established a detailed comparison between the interactions of TBP with two consensus sequences, we have now examined TBP recognition of a variant sequence. The C7 sequence (TATAAACG) is a naturally occurring single base variant of the AdMLP sequence. TBP-bound C7 has a helical bend dramatically different from that in bound AdMLP that correlates with significantly reduced relative transcription efficiency (34Wu J. Parkhurst K.M. Powell R.M. Brenowitz M. Parkhurst L.J. J. Biol. Chem. 2001; 276: 14614-14622Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). This solution geometry is highly sensitive to the presence and concentration of osmolytes (35Wu J. Parkhurst K.M. Powell R.M. Parkhurst L.J. J. Biol. Chem. 2001; 276: 14623-14627Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar), in contrast to that for the complexes bearing consensus sequences. Detailed kinetic and energetic profiles for the TBP·C7 interaction have been determined using FRET-based measurements. A comprehensive comparison with the TBP·AdMLP and TBP·E4 profiles clearly distinguishes the recognition process by TBP of the variant sequence from those of the consensus sequences. The ensemble of TBP·C7 data is well described by a three-step model with two branching pathways. One of these pathways is unique to the variant sequence mechanism and yields a bound species with DNA bent only slightly. Along the other pathway, the variant reaction proceeds through an intermediate conformer bearing strongly bent DNA in a process remarkably reminiscent of the consensus reactions. The temperature dependence of the TBP-bound C7 solution bend angle is proposed to arise from population shifts between conformers with slightly and severely bent DNA within a two-state model. Fourteen base fluorescently labeled DNA oligonucleotide probes and the unlabeled complementary strands were synthesized and purified by Sigma-Genosys (The Woodlands, TX) as described previously (16Parkhurst K.M. Richards R.M. Brenowitz M. Parkhurst L.J. J. Mol. Biol. 1999; 289: 1327-1341Crossref PubMed Scopus (72) Google Scholar, 20Powell R.M. Parkhurst K.M. Brenowitz M. Parkhurst L.J. J. Biol. Chem. 2001; 276: 29782-29791Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar, 35Wu J. Parkhurst K.M. Powell R.M. Parkhurst L.J. J. Biol. Chem. 2001; 276: 14623-14627Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). TAMRA and fluorescein were linked covalently to the 5′- and 3′-ends, respectively, via six carbon linkers to form the double-labeled C7 top strand, TAMRA-5′GGGCTATAAACGGG3′-fluorescein (duplex denoted as T*C7dpx*F) with the single-labeled oligonucleotide having only the 3′-fluorescein (duplex denoted as C7dpx*F). All duplexes were formed using a 2× excess of complement. Full-length S. cerevisiae TBP was prepared as described previously (15Parkhurst K.M. Brenowitz M. Parkhurst L.J. Biochemistry. 1996; 35: 7459-7465Crossref PubMed Scopus (119) Google Scholar, 18Petri V. Hsieh M. Brenowitz M. Biochemistry. 1995; 34: 9977-9984Crossref PubMed Scopus (89) Google Scholar). Studies were conducted in 10 mm Tris-HCl (pH 7.4), 100 mm KCl, 2.5 mm MgCl2, 1 mm CaCl2and 1 mm dithiothreitol at the temperatures indicated. Determination of equilibrium isotherms at 15, 20, 25, and 30 °C is described in the legend of Fig. 1. The interaction of TBP with T*C7dpx*F was monitored using fluorescence resonance energy transfer (FRET) together with various fluorescence techniques. Detailed discussions of FRET and its applications to the present study have been published (15Parkhurst K.M. Brenowitz M. Parkhurst L.J. Biochemistry. 1996; 35: 7459-7465Crossref PubMed Scopus (119) Google Scholar, 16Parkhurst K.M. Richards R.M. Brenowitz M. Parkhurst L.J. J. Mol. Biol. 1999; 289: 1327-1341Crossref PubMed Scopus (72) Google Scholar, 34Wu J. Parkhurst K.M. Powell R.M. Brenowitz M. Parkhurst L.J. J. Biol. Chem. 2001; 276: 14614-14622Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar,36Cheung H.C. Lakowicz J.R. Topics in Fluorescence Spectroscopy. Plenum Press, New York1991: 157-171Google Scholar, 37Lakowicz J.R. Principles of Fluorescence Spectroscopy.2nd Ed. Plenum Publishers, New York1999: 368-442Google Scholar, 38Parkhurst L.J. Parkhurst K.M. Proc. Soc. Photo-Optical Instrum. Eng. 1994; 2137: 475-483Google Scholar, 39Parkhurst K.M. Parkhurst L.J. Biochemistry. 1995; 34: 293-300Crossref PubMed Scopus (74) Google Scholar, 40Parkhurst K.M. Parkhurst L.J. Biochemistry. 1995; 34: 285-292Crossref PubMed Scopus (96) Google Scholar, 41Wu P. Brand L. Anal. Biochem. 1994; 218: 1-13Crossref PubMed Scopus (1122) Google Scholar, 42Wu P. Brand L. Biochemistry. 1992; 31: 7939-7947Crossref PubMed Scopus (116) Google Scholar). Briefly, the rate constant for the non-radiative transfer of excited state energy from a donor to an acceptor fluorophore is highly dependent upon the distance separating the two dyes. Because the binding of TBP to a TATA-bearing promoter induces a bend in the DNA helical axis, binding to T*C7dpx*F results in a decrease in the 5′TAMRA-3′ fluorescein distance. The corresponding change in the efficiency of energy transfer is reflected in the fluorescence emission of the dyes and may be used to monitor these interactions with TBP in real time. For non-rigid molecules such as the fluorescently labeled oligomeric probes used in these studies, the 5′-end to 3′-end distance is described by a probability distribution, P(R), rather than by a single fixed value. Such distributions were extracted from time-resolved fluorescence emission measurements of free and TBP-bound T*C7dpx*F at 15, 20, 25, and 30 °C as described (34Wu J. Parkhurst K.M. Powell R.M. Brenowitz M. Parkhurst L.J. J. Biol. Chem. 2001; 276: 14614-14622Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). Model-dependent bend angles for the bound duplex were then determined as described (34Wu J. Parkhurst K.M. Powell R.M. Brenowitz M. Parkhurst L.J. J. Biol. Chem. 2001; 276: 14614-14622Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 35Wu J. Parkhurst K.M. Powell R.M. Parkhurst L.J. J. Biol. Chem. 2001; 276: 14623-14627Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). The association binding of TBP with T*C7dpx*F has been investigated as described for TBP binding the adenovirus major late (AdMLP) and E4 promoters (16Parkhurst K.M. Richards R.M. Brenowitz M. Parkhurst L.J. J. Mol. Biol. 1999; 289: 1327-1341Crossref PubMed Scopus (72) Google Scholar, 20Powell R.M. Parkhurst K.M. Brenowitz M. Parkhurst L.J. J. Biol. Chem. 2001; 276: 29782-29791Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar) using stopped-flow fluorimetry. Briefly, solutions of 40 nm T*C7dpx*F were flowed against TBP solutions of 400, 800, or 1600 nm (prior to mixing), using a 1:1 mixing ratio, at 15, 20, 25, and 30 °C. Averaged replicate curves were fit using exponential decay models. The resulting set of eight curves, each deriving from a unique combination of temperature and TBP concentration, was used in global analyses. TBP·T*C7dpx*F complexes were challenged with a large excess of unlabeled C7 duplex (C7dpx), and dissociation was monitored using steady-state FRET as described (16Parkhurst K.M. Richards R.M. Brenowitz M. Parkhurst L.J. J. Mol. Biol. 1999; 289: 1327-1341Crossref PubMed Scopus (72) Google Scholar,20Powell R.M. Parkhurst K.M. Brenowitz M. Parkhurst L.J. J. Biol. Chem. 2001; 276: 29782-29791Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). The initial complex incorporating labeled DNA was formed and equilibrated using 19.2–20.3 nm T*C7dpx*F and 992.3–1004 nm TBP, the latter sufficient to ensure ≥94% duplex saturation at all temperatures. Dissociation kinetic data were obtained as a function of unlabeled DNA concentration, with C7dpx added to final concentrations of 17.9, 24.8, or 35.5 μm. Data obtained at 15, 20, and 25 °C were all well fit using a biexponential decay model as described (16Parkhurst K.M. Richards R.M. Brenowitz M. Parkhurst L.J. J. Mol. Biol. 1999; 289: 1327-1341Crossref PubMed Scopus (72) Google Scholar, 20Powell R.M. Parkhurst K.M. Brenowitz M. Parkhurst L.J. J. Biol. Chem. 2001; 276: 29782-29791Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). A dependence of the TBP·T*C7dpx*F dissociation kinetics on C7dpx concentration was observed, indicating two contributions to the dissociation process: first-order replacement of labeled by unlabeled duplex and active removal, or second-order displacement, of labeled duplex. Similar complexity has been reported for TBP·T*E4dpx*F dissociation kinetics (20Powell R.M. Parkhurst K.M. Brenowitz M. Parkhurst L.J. J. Biol. Chem. 2001; 276: 29782-29791Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). Extraction of the pure replacement curve at each temperature was necessary, because only that process is considered in subsequent global analysis. Two model-independent methods (20Powell R.M. Parkhurst K.M. Brenowitz M. Parkhurst L.J. J. Biol. Chem. 2001; 276: 29782-29791Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar) were used for this procedure. The recovered replacement kinetic curves at 15, 20, and 25 °C were subsequently used in the global analyses. The eight stopped-flow binding and three replacement kinetic curves and four equilibrium binding isotherms characterizing the TBP·C7 interaction were analyzed globally to explore their correspondence with various kinetic models. Analyses based on two- and three-step models, including both linear and branching schemes, are described below. To ensure meaningful comparisons with the previously determined results for TBP binding to consensus sequences, analyses were conducted in a manner analogous to those for TBP·AdMLP and TBP·E4 (16Parkhurst K.M. Richards R.M. Brenowitz M. Parkhurst L.J. J. Mol. Biol. 1999; 289: 1327-1341Crossref PubMed Scopus (72) Google Scholar, 20Powell R.M. Parkhurst K.M. Brenowitz M. Parkhurst L.J. J. Biol. Chem. 2001; 276: 29782-29791Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). Smoothed representations of the association and replacement curves and equilibrium binding isotherms were constructed as described (16Parkhurst K.M. Richards R.M. Brenowitz M. Parkhurst L.J. J. Mol. Biol. 1999; 289: 1327-1341Crossref PubMed Scopus (72) Google Scholar, 20Powell R.M. Parkhurst K.M. Brenowitz M. Parkhurst L.J. J. Biol. Chem. 2001; 276: 29782-29791Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). The latter were scaled from 0 to 0.127 (15 °C), 0.216 (20 °C), 0.301 (25 °C), or 0.335 (30 °C), the temperature-dependent amplitude changes observed upon TBP·T*C7dpx*F binding in steady-state FRET measurements. The theoretical response functions were determined as described (16Parkhurst K.M. Richards R.M. Brenowitz M. Parkhurst L.J. J. Mol. Biol. 1999; 289: 1327-1341Crossref PubMed Scopus (72) Google Scholar,20Powell R.M. Parkhurst K.M. Brenowitz M. Parkhurst L.J. J. Biol. Chem. 2001; 276: 29782-29791Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). The overall average squared residual, ςglobal2, derived from the weighted variance between the observed and theoretical points describing the shapes of the association (ςSF2), replacement (ςR2), and equilibrium binding (ςK2) curves,ςglobal2=113[8ςSF2+2ςK2+3ςR2]Equation 1 with the coefficients reflecting the relative information content of each term. A fit with each term within its experimental error thus yields a value for ςglobal2 ≤ 1. Error estimates for the optimal parameter values were obtained exactly as described (16Parkhurst K.M. Richards R.M. Brenowitz M. Parkhurst L.J. J. Mol. Biol. 1999; 289: 1327-1341Crossref PubMed Scopus (72) Google Scholar). The correspondence of the ensemble of TBP·C7 data was first tested against linear (Equation 2) and branching (Equation 3) two-step models, where the subscript “INT” denotes an intermediate conformer. Global analysis for either model yielded values for the four rate constants at 30 °C as well as the corresponding activation enthalpies. The quantum yields of the donor fluorescein in the two TBP-bound duplexes, relative to that of free T*C7dpx*F, were also determined. These quantum yield values indicate the relative extent of DNA bending in each binary complex, as they reflect the 5′dye-3′dye distance (15Parkhurst K.M. Brenowitz M. Parkhurst L.J. Biochemistry. 1996; 35: 7459-7465Crossref PubMed Scopus (119) Google Scholar, 16Parkhurst K.M. Richards R.M. Brenowitz M. Parkhurst L.J. J. Mol. Biol. 1999; 289: 1327-1341Crossref PubMed Scopus (72) Google Scholar, 34Wu J. Parkhurst K.M. Powell R.M. Brenowitz M. Parkhurst L.J. J. Biol. Chem. 2001; 276: 14614-14622Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). The data were further tested against various three-step mechanisms. First explored was a linear two-intermediate kinetic model, shown previously to describe both the TBP·AdMLP and TBP·E4 reactions (16Parkhurst K.M. Richards R.M. Brenowitz M. Parkhurst L.J. J. Mol. Biol. 1999; 289: 1327-1341Crossref PubMed Scopus (72) Google Scholar, 20Powell R.M. Parkhurst K.M. Brenowitz M. Parkhurst L.J. J. Biol. Chem. 2001; 276: 29782-29791Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar). Determined in the analysis were values for the six rate constants at 30 °C, the corresponding activation energies, and the quantum yield for each TBP-bound conformer relative to that of the free duplex. To reduce the number of fitting parameters, additional analyses were conducted with quantum yields constrained to be equivalent for either conformers A and B or conformers B and C, consistent with a two-state model for this interaction (34Wu J. Parkhurst K.M. Powell R.M. Brenowitz M. Parkhurst L.J. J. Biol. Chem. 2001; 276: 14614-14622Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 35Wu J. Parkhurst K.M. Powell R.M. Parkhurst L.J. J. Biol. Chem. 2001; 276: 14623-14627Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar). Two additional three-step models were explored, with two and three branching pathways, with the rate constants k 2 andk 3 in Equation 5 and k 2,k 3, and k 5 in Equation 6 being second-order. Relative quantum yield values were determined for each TBP-bound conformer, and additional analyses conducted with constraints on the quantum yields as for Equation 4. Finally, Equation 5 was extended to allow direct conversion between TBP·DNAA and TBP·DNAB, INT via rate constants k 7 and k 8. Numerous independent studies have demonstrated that our preparations of yeast TBP are monomeric and thermally stable under the conditions of the studies described herein (16Parkhurst K.M. Richards R.M. Brenowitz M. Parkhurst L.J. J. Mol. Biol. 1999; 289: 1327-1341Crossref PubMed Scopus (72) Google Scholar, 20Powell R.M. Parkhurst K.M. Brenowitz M. Parkhurst L.J. J. Biol. Chem. 2001; 276: 29782-29791Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar, 34Wu J. Parkhurst K.M. Powell R.M. Brenowitz M. Parkhurst L.J. J. Biol. Chem. 2001; 276: 14614-14622Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 43Campbell K.M. Ranallo R.T. Stargell L.A. Lumb K.J. Biochemistry. 2000; 39: 2633-2638Crossref PubMed Scopus (27) Google Scholar, 44Daugherty M.A. Brenowitz M. Fried M.G. J. Mol. Biol. 1999; 285: 1389-1399Crossref PubMed Scopus (30) Google Scholar, 45Daugherty M.A. Brenowitz M. Fried M.G. Biochemistry. 2000; 39: 4869-4880Crossref PubMed Scopus (21) Google Scholar). Four equilibrium isotherms for TBP·T*C7dpx*F binding were obtained using steady-state FRET from 15 to 30 °C, shown for 25 °C in Fig.1. These data were well fit by linear regression using the van't Hoff equation, yieldingK a values, in μm−1, of 10.3 ± 5.0 at 15 °C, 17.2 ± 5.3 at 20 °C, 28.2 ± 8.7 at 25 °C, and 45.4 ± 20.3" @default.
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• W2002899891 title "Comparison of TATA-binding Protein Recognition of a Variant and Consensus DNA Promoters" @default.
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