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• W2044804986 abstract "High mobility protein-1 (HMG-1) has been shown to regulate transcription by RNA polymerase II. In the context that it acts as a transcriptional repressor, it binds to the TATA-binding protein (TBP) to form the HMG-1/TBP/TATA complex, which is proposed to inhibit the assembly of the preinitiation complex. By using electrophoretic mobility shift assays, we show that the acidic C-terminal domain of HMG-1 and the N terminus of human TBP are the domains that are essential for the formation of a stable HMG-1/TBP/TATA complex. HMG-1 binding increases the affinity of TBP for the TATA element by 20-fold, which is reflected in a significant stimulation of the rate of TBP binding, with little effect on the dissociation rate constant. In support of the binding target of HMG-1 being the N terminus of hTBP, the N-terminal polypeptide of human TBP competes with and inhibits HMG-1/TBP/TATA complex formation. Deletion of segments of the N terminus of human TBP was used to map the region(s) where HMG-1 binds. These findings indicate that interaction of HMG-1 with the Q-tract (amino acids 55–95) in hTBP is primarily responsible for stable complex formation. In addition, HMG-1 and the monoclonal antibody, 1C2, specific to the Q-tract, compete for the same site. Furthermore, calf thymus HMG-1 forms a stable complex with the TBP/TATA complex that contains TBP from either human or Drosophilabut not yeast. This is again consistent with the importance of the Q-tract for this stable interaction and shows that the interaction extends over many species but does not include yeast TBP. High mobility protein-1 (HMG-1) has been shown to regulate transcription by RNA polymerase II. In the context that it acts as a transcriptional repressor, it binds to the TATA-binding protein (TBP) to form the HMG-1/TBP/TATA complex, which is proposed to inhibit the assembly of the preinitiation complex. By using electrophoretic mobility shift assays, we show that the acidic C-terminal domain of HMG-1 and the N terminus of human TBP are the domains that are essential for the formation of a stable HMG-1/TBP/TATA complex. HMG-1 binding increases the affinity of TBP for the TATA element by 20-fold, which is reflected in a significant stimulation of the rate of TBP binding, with little effect on the dissociation rate constant. In support of the binding target of HMG-1 being the N terminus of hTBP, the N-terminal polypeptide of human TBP competes with and inhibits HMG-1/TBP/TATA complex formation. Deletion of segments of the N terminus of human TBP was used to map the region(s) where HMG-1 binds. These findings indicate that interaction of HMG-1 with the Q-tract (amino acids 55–95) in hTBP is primarily responsible for stable complex formation. In addition, HMG-1 and the monoclonal antibody, 1C2, specific to the Q-tract, compete for the same site. Furthermore, calf thymus HMG-1 forms a stable complex with the TBP/TATA complex that contains TBP from either human or Drosophilabut not yeast. This is again consistent with the importance of the Q-tract for this stable interaction and shows that the interaction extends over many species but does not include yeast TBP. TATA-binding protein adenovirus major late promoter electrophoretic mobility shift assay glutathione S-transferase high mobility group-1 protein human TATA-binding protein monoclonal antibody glutamine small nuclear RNA-activating protein complex polymerase the C terminus of human TBP, residues 160-335 the N terminus of human TBP, residues 1-159 Drosophila TBP yeast TBP The TATA-binding protein is a universal transcription factor that is essential for eukaryotic transcription by all three RNA polymerases (1Hernandez N. Genes Dev. 1993; 7: 1291-1308Crossref PubMed Scopus (563) Google Scholar, 2Burley S.K. Roeder R.G. Annu. Rev. Biochem. 1996; 65: 769-799Crossref PubMed Scopus (623) Google Scholar, 3Nikolov D.B. Burley S.K. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 15-22Crossref PubMed Scopus (187) Google Scholar, 4Nikolov D.B. Burley S.K. Struct. Biol. 1994; 1: 621-637Crossref PubMed Scopus (99) Google Scholar). For RNA polymerase II transcription, the regulation of TBP1 binding to the TATA element is considered a principal determinant in promoter activity and therefore a primary target for regulatory factors. TBP can be considered modular in nature, with its highly conserved C terminus being necessary and sufficient for both binding to the TATA box and basal level transcription (5Hoey T. Weinzierl R.O.J. Gill G. Chen J.L. Dynlacht B.D. Tjian R. Cell. 1993; 72: 247-260Abstract Full Text PDF PubMed Scopus (475) Google Scholar, 6Horikoshi M. Yamamoto T. Okhuma Y. Weil P.A. Roeder R.G. Cell. 1990; 61: 1171-1178Abstract Full Text PDF PubMed Scopus (101) Google Scholar). In addition all activators and repressors that bind to human TBP (hTBP) are reported to bind to the C terminus (2Burley S.K. Roeder R.G. Annu. Rev. Biochem. 1996; 65: 769-799Crossref PubMed Scopus (623) Google Scholar, 3Nikolov D.B. Burley S.K. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 15-22Crossref PubMed Scopus (187) Google Scholar, 4Nikolov D.B. Burley S.K. Struct. Biol. 1994; 1: 621-637Crossref PubMed Scopus (99) Google Scholar). On the other hand, the interactions of regulatory factors with the 159-residue N terminus in hTBP appear much more limited, and its role in transcriptional regulation is not understood. In the only case that is characterized, it was shown that the N terminus down-regulates hTBP binding to the U6 TATA box, mediates cooperative binding with SNAPc to the U6 promoter, and facilitates an enhanced level of RNA polymerase III transcription of the U6gene (7Mittal V. Hernandez N. Science. 1997; 275: 1136-1140Crossref PubMed Scopus (77) Google Scholar, 8Henry R.W. Ford E. Mital R. Mittal V. Hernandez N. Cold Spring Harbor Symp. Quant. Biol. 1998; 63: 111-120Crossref PubMed Scopus (32) Google Scholar). In addition, a monoclonal antibody specific for the Q-tract of the N terminus of hTBP was shown to inhibit selectivelyin vitro transcription from TATA-containing, but not TATA-less, promoters that were transcribed by RNA pol II or III. This antibody interaction did not affect TBP binding to the TATA box or inhibit the formation of the TFIIA/TFIIB/TBP/TATA complex, which suggests that the N terminus may be available for protein-protein interactions associated with subsequent assembly of the preinitiation complex (9Lescure A. Lutz Y. Eberhard D. Jacq X. Krol A. Grummt I. Davidson I. Chambon P. Tora L. EMBO J. 1994; 13: 1166-1175Crossref PubMed Scopus (94) Google Scholar). HMG-1 is a ubiquitous and highly conserved nuclear protein that has been reported to serve as a transcriptional repressor (10Ge H. Roeder R.G. J. Biol. Chem. 1994; 269: 17136-17140Abstract Full Text PDF PubMed Google Scholar, 11Stelzer G. Goppelt A. Lottspeich F. Meisterernst M. Mol. Cell. Biol. 1994; 14: 4712-4721Crossref PubMed Scopus (55) Google Scholar) in some systems, while functioning as a coactivator for RNA polymerase II in other contexts (12Onate S. Prendergast P. Wagner J. Nissen M. Reeves R. Pettijohn D. Edwards D. Mol. Cell. Biol. 1994; 14: 3376-3391Crossref PubMed Google Scholar, 13Verrier C.S. Roodi N. Yee C.J. Bailey L.R. Jensen R.A. Bustin M. Parl F.F. Mol. Endocrinol. 1997; 11: 1009-1019Crossref PubMed Scopus (71) Google Scholar, 14Boonyaratanakornkit V. Melvin V. Prendergast P. Altmann M. Ronfani L. Bianchi M. Taraseviciene L. Nordenn S.K. Allergretto E. Edwards D. Mol. Cell. Biol. 1998; 18: 4471-4487Crossref PubMed Scopus (304) Google Scholar, 15Zappavigna V. Falciola L. Citterich M. Mavilio F. Bianchi M.E. EMBO J. 1996; 15: 4981-4991Crossref PubMed Scopus (216) Google Scholar, 16Jayaraman L. Moorthy N.C. Murthy K.G. Manley J.L. Bustin M. Prives C. Genes Dev. 1998; 12: 462-472Crossref PubMed Scopus (281) Google Scholar, 17Ellwood K.B. Yen Y.-M. Johnson R.C. Carey M. Mol. Cell. Biol. 2000; 20: 4359-4370Crossref PubMed Scopus (81) Google Scholar). Fig. 1A shows that HMG-1 is likewise modular in nature, consisting of three domains. The A- and B-domains, each containing about 80 residues with a high percentage of arginines and lysines, are homologous and structurally comparable and have been shown to bind nonspecifically to DNA (18Bustin M. Mol. Cell. Biol. 1999; 19: 5237-5246Crossref PubMed Scopus (754) Google Scholar, 19Bustin M. Reeves R. Prog. Nucleic Acid Res. Mol. Biol. 1996; 54: 35-99Crossref PubMed Google Scholar, 20Bustin M. Lehn D.A. Landsman D. Biochim. Biophys. Acta. 1990; 1049: 231-243Crossref PubMed Scopus (438) Google Scholar, 21Landsman D. Bustin M. BioEssays. 1993; 15: 539-546Crossref PubMed Scopus (237) Google Scholar). The C terminus is quite different, being polyanionic, with the last 30 residues being a stretch of exclusively aspartic or glutamic acid residues. This segment reduces binding affinity to DNA and is not required for protein stability (22Bianchi M. Falciola L. Ferrari S. Lilley D. EMBO J. 1992; 11: 1055-1063Crossref PubMed Scopus (214) Google Scholar) but has defied a more definitive functional role. In this work, we show that the C terminus of HMG-1 and the Q-tract in the N terminus of human TBP are essential for stable HMG-1/TBP/TATA complex formation. HMG-1 increases the affinity of TBP for the TATA element 20-fold, which is reflected in a significant increase in the rate of TBP binding, while having little effect on the lifetime of the complex. This interaction provides a broader spectrum of regulatory controls for TBP binding and promoter activity. Calf thymus HMG-1 and HMG-2 proteins were purified in non-denaturing conditions using salt extraction, selective precipitation with ammonium sulfate, and further purification by high pressure liquid chromatography using a MonoQ column (23Marekov L.N. Demirov D.G. Beltchev B.G. Biochim. Biophys. Acta. 1984; 789: 63-68Crossref PubMed Scopus (24) Google Scholar, 24Lu W. Peterson R. Dasgupta A. Scovell W.M. J. Biol. Chem. 2000; 275: 35006-35012Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar). The expression vector for HMG-1(A-B) didomain (residues 1–176), obtained from M. Bianchi, was transfected into BL21(DE3) cells, and the expressed protein was purified using published protocols (25Stros M. Reich J. Kolibalova A. FEBS Lett. 1994; 344: 201-206Crossref PubMed Scopus (40) Google Scholar). The expression vectors, pET-his6hTBP, pET11d-his6180hcTBP (from F. Pugh), pAR3038dTBP (from R. Tjian), and pET11d-his6yTBP (from R. Roeder), were transfected into BL21(DE3) cells, and the expressed proteins were purified using phosphocellulose chromatography and ammonium sulfate precipitation, as described by Pugh (26Pugh F. Methods Mol. Biol. 1995; 37: 359-368PubMed Google Scholar). The GST fusion proteins with full-length TBP or TBP with N-terminal deletions (ΔN) (from N. Hernandez) were obtained as pET11c expression vectors (7Mittal V. Hernandez N. Science. 1997; 275: 1136-1140Crossref PubMed Scopus (77) Google Scholar), transfected into BL21(DE3) cells, and expressed and purified using glutathione-agarose (Sigma) (7Mittal V. Hernandez N. Science. 1997; 275: 1136-1140Crossref PubMed Scopus (77) Google Scholar). The GST-nTBP-(1–158) (from T. Kouzarides) was purified according to standard procedures and was used in competition experiments. The purity of all proteins isolated was >90% as evidenced by a single Coomassie-stained band on SDS-PAGE. The individual oligonucleotides of the adenovirus major late promoter (AdMLP; −40 to −1 and −1 to −40) were purchased from National Biosystems and32P-end-labeled by standard procedures (27Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. 2nd Ed. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). The EMSA procedures have been described (24Lu W. Peterson R. Dasgupta A. Scovell W.M. J. Biol. Chem. 2000; 275: 35006-35012Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar) but involved reacting the DNA probe with the proteins of interest in binding buffer (24 mm Tris acetate, pH 8.0, 10% glycerol, 4 mm magnesium acetate, 50 mm potassium glutamate, 0.1 mm EDTA, 1 mm dithiothreitol, 0.01% Nonidet P-40, 4 mmspermidine, 5 µg/ml poly(dG-dC), and 115 µg/µl bovine serum albumin) for 30 min at 30 °C. All samples were electrophoresed in 0.35× TBE buffer (TBE, Tris borate-EDTA buffer) at 200 V for ∼1.5 h in 4–6% nondenaturing polyacrylamide gels at 4 °C. After completion of electrophoresis, the gels were dried and exposed to x-ray film. Antibodies used in the supershift experiments were obtained from R. Roeder (anti-HMG-1) and P. Chambon (monoclonal TBP antibodies (1C2 and 3G3)). The K d values were obtained by titration of 100 pm DNA over a range of TBP concentrations, with equilibrium established after 60 min at 30 °C. Experiments for the HMG-1/TBP/TATA complex were done with the HMG-1 concentration at 120 nm. The band intensities for the complex and free DNA in these studies and those in the kinetic determinations (below) were measured by exposing the dried gels to a PhosphorImager screen, which were scanned using the Molecular Dynamics PhosphorImager system. The ImageQuant software program was used to measure accurately the band intensities. The K d value for the TBP/TATA complex is equal to the free TBP concentration at which there are equal concentrations of [DNA] and [TBP/DNA] (i.e. K d = [TBP][DNA]/[[TBP/DNA]). At the very low TBP concentrations, in which the TBP concentration was not in large excess relative to DNA, the [TBP]free was calculated by standard procedures. The fraction of complex was plottedversus the concentration of TBP to generate the binding curves. The best fit of the data was derived (using Sigma Plot for PC) using over 50 data points from five independent determinations. The off-rate constants for the complexes were determined by establishing the complex for 60 min and challenging the complex with 20 ng/µl poly(dI-dC)·poly(dI-dC). At the time points indicated, the samples were loaded on the gel. The same level of poly(dI-dC)·poly(dI-dC) was added to the controls at the corresponding time points. Reactions were initiated at staggered intervals so that all samples could be loaded on the gel at the same time. DNA concentrations were at 100 pm, with TBP concentrations being at least 15-fold in excess. The HMG-1 concentration was 120 nm. The τ 12 values were derived from the linear plot of ln[c/co] versustime, with the k off obtained from the relationship, k off = ln 2/τ 12. The relative on-rates were estimated from a plot of the fraction complex formed versus time. These plots were also derived from more than 50 points from five independent runs, with the average values used to obtain the final plot. The gel electrophoresis for both the thermodynamic and kinetic studies was run for only 20 min to minimize any dissociation of the complex during electrophoresis. HMG-1 binds to TBP/TATA to form an EMSA-stable HMG-1/TBP/TATA complex, exhibiting a significantly greater stability than the TBP/TATA complex (24Lu W. Peterson R. Dasgupta A. Scovell W.M. J. Biol. Chem. 2000; 275: 35006-35012Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar). To investigate the extent to which the acidic C terminus of HMG-1 contributes to the stability of the complex, the binding of HMG-1-(1–215) to TBP was compared with that for the didomain HMG-1(A-B)-(1–176) that lacks the C terminus. The EMSA profile in Fig.1 B compares the binding of HMG-1 (lanes 3–7) and HMG-1(A-B) (lanes 9–14) to the TBP/TATA complex. HMG-1 binding produces a complex with an increased mobility, as reported previously (24Lu W. Peterson R. Dasgupta A. Scovell W.M. J. Biol. Chem. 2000; 275: 35006-35012Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar). The HMG-1/TBP/TATA complex is evident at an (HMG-1/TBP) molar ratio of 4, whereas it is the sole species at a molar ratio of 40. On the other hand, no complex is detectable with HMG-1(A-B) at a molar ratio as high as 640. A strong new band of increased mobility becomes apparent at these increasingly higher HMG-1(A-B) levels (lanes 11–14).Lanes 15–18 confirm that this major band observed inlanes 11–14 is also observed when HMG-1(A-B) is reacted with the DNA probe in the absence of TBP. This indicates that at HMG-1(A-B) molar ratios of 80 or greater, the highly charged HMG-1(A-B) binds directly and preferentially to DNA and not to the TBP. This is consistent with the much higher DNA binding affinity expected for HMG-1(A-B) compared with HMG-1 (28Stros M. Stokrova J. Thomas J.O. Nucleic Acids Res. 1994; 22: 1044-1051Crossref PubMed Scopus (144) Google Scholar, 29Sheflin L.G. Fucile N.W. Spaulding S.W. Biochemistry. 1993; 32: 3238-3248Crossref PubMed Scopus (115) Google Scholar, 30Grasser K.D. Teo S.H. Lee K.B. Broadhurst W. Rees C. Hardman C.H. Thomas J.O. Eur. J. Biochem. 1998; 253: 787-795Crossref PubMed Scopus (72) Google Scholar). We conclude that the C terminus is essential for the stability of the HMG-1/TBP/TATA complex. The role of the N terminus of hTBP was examined by comparing the relative binding of HMG-1 to both the full-length hTBP-(1–339) and the C-terminal TBP fragment (residues 159–339).Lanes 1–6 in Fig. 2, like the data in Fig. 1, show the strong binding profile for HMG-1 to TBP/TATA, with complete complex formation at a (HMG-1/TBP) molar ratio of 40. However, incubation of HMG-1 with cTBP/TATA complex does not lead to any detectable complex formation, as evident in lanes 9–14.Complexation of HMG-1 with cTBP/TATA could not be detected at (HMG-1/cTBP) molar ratios as high as 640. In fact, at ratios of about 200 and higher, HMG-1 inhibits cTBP binding to the TATA-containing probe. Together with this, a band of greater mobility is observed again, which results from HMG-1 binding directly and nonspecifically to the DNA probe. This interpretation is verified by reacting HMG-1 with the DNA probe in the absence of cTBP (lanes 15–17) which produces the same band profile. The relative stabilities of the TBP/TATA and HMG-1/TBP/TATA complexes were quantitatively compared by titrating the TATA-containing oligonucleotide with TBP, in the absence and presence of saturating levels of HMG-1. The EMSA binding profiles for the two complexes are shown in Fig.3A. Qualitative examination of the binding at low TBP levels (compare lanes 2 and11) shows more complex formed in the presence of HMG-1. The band intensity data were used to plot the fraction of each complex formed as a function of TBP concentration (Fig. 3 B), from which the corresponding K d values were determined. The K d value for the TBP/TATA complex was 1.5 nm, which is comparable to values reported previously (31Hoopes B.C. LeBlanc J.F. Hawley D.K. J. Biol. Chem. 1992; 267: 11539-11547Abstract Full Text PDF PubMed Google Scholar, 32Weideman C.A. Netter R.C. Benjamin L.R. McAllister J.J. Schmiedekamp R.A. Coleman R.A. Pugh B.F. J. Mol. Biol. 1997; 271: 61-75Crossref PubMed Scopus (47) Google Scholar, 33Wolner B.S. Gralla J.D. J. Biol. Chem. 2001; 276: 6260-6266Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar, 34Imbalzano A.N. Zaret K.S. Kingston R.E. J. Biol. Chem. 1994; 269: 8280-8286Abstract Full Text PDF PubMed Google Scholar). The corresponding plot for TBP binding in the presence of saturating levels of HMG-1 shows stimulated binding of TBP to the TATA element. Complex formation is observed at significantly lower TBP levels than required for the binding of only TBP to the TATA element. This complexation reduced the K d value by about 20-fold, with 50% HMG-1/TBP/TATA complex formation occurring at about 70 pm TBP. HMG-2 protein is similar to HMG-1 in size and exhibits a high degree of homology, with both proteins being implicated in the regulation of transcription (18Bustin M. Mol. Cell. Biol. 1999; 19: 5237-5246Crossref PubMed Scopus (754) Google Scholar, 19Bustin M. Reeves R. Prog. Nucleic Acid Res. Mol. Biol. 1996; 54: 35-99Crossref PubMed Google Scholar, 20Bustin M. Lehn D.A. Landsman D. Biochim. Biophys. Acta. 1990; 1049: 231-243Crossref PubMed Scopus (438) Google Scholar, 21Landsman D. Bustin M. BioEssays. 1993; 15: 539-546Crossref PubMed Scopus (237) Google Scholar). However, HMG-2 has eight fewer acidic residues (22 versus 30 in HMG-1) in the C-terminal acidic tract (35Wen L. Huang J.K. Johnson B.H. Reeck G.R. Nucleic Acids Res. 1989; 27: 1197-1214Crossref Scopus (154) Google Scholar,36Majumdar A. Brown D. Kerby S. Rudzinski I. Polte T. Randhawa Z. Seidman M. Nucleic Acids Res. 1991; 19: 6643Crossref PubMed Scopus (24) Google Scholar). We determined that HMG-2 also stimulates TBP binding, with both proteins exhibiting comparable effects in enhancing TBP binding (data not shown). Since the K d value is a reflection of the ratio ofk d/k on, the impact of HMG-1 on the complex dissociation rate constant was determined. The EMSA profile for dissociation for each complex is shown in Fig.4A, with the data plotted in Fig. 4 B. The dissociation profiles for the complexes differ only slightly, indicating that the presence of HMG-1 has little effect on the half-life of the complex. The values of τ 12 are 160 and 130 min, respectively, for the TBP/TATA and the HMG-1/TBP/TATA complexes. The corresponding values for k off are 7.2 × 10−5 and 8.9 × 10−5s−1, respectively. The value for the TBP/TATA complex is comparable to previously reported values (33Wolner B.S. Gralla J.D. J. Biol. Chem. 2001; 276: 6260-6266Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar) obtained for TBP dissociation. This finding would indicate that the effect of HMG-1 on theK d value should be associated predominantly with an increase in the on-rate for TBP. To obtain an estimate of the relative on-rates and determine if this was generally consistent with theK d data, the comparative time course of TBP binding was monitored for the two complexes and is shown in Fig. 4 C. Comparison of the relative band intensities at the same time points (e.g. 5 min) shows that, qualitatively, the presence of HMG-1 stimulates the rate of TBP binding. As shown in Fig.4 D, quantitative measurements indicate that HMG-1 clearly stimulates the rate of TBP binding, enhancing the initial rate by about 10-fold. The initial slope for the formation of the HMG-1/TBP/TATA complex represents only an estimate or lower limit value due to the high rate of reaction and difficulty of obtaining consistent data at times less than 1 min. These kinetic data are, however, consistent with the thermodynamic data and indicate that HMG-1 decreases theK d value by primarily increasing the on-rate kinetics, while having little discernible effect on the dissociation kinetics. It was of interest to provide additional support for the role of the N terminus of TBP as the target for HMG-1. If HMG-1 interacts directly with the N terminus of hTBP and this provides the primary stability for the complex, then the presence of the exogenous N-terminal polypeptide would be expected to inhibit the formation of the HMG-1/TBP/TATA complex. Fig. 5shows the effect of increasing levels of GST-nTBP-(1–159), 34–260 nm (lanes 2–6), when it is preincubated with 80 nm HMG-1 for 20 min on ice, followed by 30 min of incubation with TBP/TATA. Little inhibition is observed at the lower level of nTBP (molar ratio (nTBP/HMG-1) of 0.5; (nTBP/TBP) of 34) (lane 2), whereas progressive inhibition occurs at the higher nTBP levels, with complete inhibition of complex formation observed at about the 225 nm level (molar ratio (nTBP/HMG-1) of 3; (nTBP/TBP) of 225) (lane 4–5). The presence of nTBP exhibited no detectable effect on TBP/TATA complex formation (data not shown). These data provide additional support for the N terminus of hTBP as the principal target for HMG-1 binding and for interaction with this region providing the primary stability in complex formation. In order to determine if a particular segment of the N terminus of hTBP might play a predominant role in the HMG-1 interaction, the effect that HMG-1 has on increasing the stability of the TBP/TATA complex was examined using hTBP deletion mutants. The schematic of hTBP and the five N-terminal deletion mutants used are shown in Fig. 6A. The non-conserved 159-residue N terminus can be conveniently divided into three regions as follows: the segment containing the initial residues from 1 to 54 (fragment I); the central region, inclusive of residues 55–95 and containing the Q-tract, which is made up of 34 consecutive glutamine residues (fragment II); and the segment from 96 to 158 (fragment III), which lies between the Q-tract and the conserved C terminus (residues 159–335). The ΔN nomenclature for the mutants is from Mittal and Hernandez (7Mittal V. Hernandez N. Science. 1997; 275: 1136-1140Crossref PubMed Scopus (77) Google Scholar). The pairs of adjacent lanes in Fig.6 B show the relative stability of the TBP/TATA and the corresponding HMG-1/TBP/TATA complexes. Comparison of lanes 1 and 2 shows that the extent of GST-TBP binding to TATA (lane 1) is significantly increased by complexation with HMG-1. This is essentially the same value obtained when TBP (not in the GST fusion) is used. This indicates that the presence of the GST does not change the HMG-1/TBP binding, which is also what was observed previously with the SNAPc/TBP binding (7Mittal V. Hernandez N. Science. 1997; 275: 1136-1140Crossref PubMed Scopus (77) Google Scholar, 8Henry R.W. Ford E. Mital R. Mittal V. Hernandez N. Cold Spring Harbor Symp. Quant. Biol. 1998; 63: 111-120Crossref PubMed Scopus (32) Google Scholar). The relative increases for the mutant-TBPs were determined using a PhosphorImager (data not shown) and were compared with this value. The mutants were found to fall into two different groups. HMG-1 increases the stability of the complex formed for both ΔN + I + II (lanes 3 and4) and ΔN + II (lanes 5 and 6), with the stability being comparable, but slightly less, than that for hTBP itself. This suggests that segment II (the Q-tract) plays the major role in stabilizing the interaction with HMG-1, with segment I providing little additional stability. Fragment ΔN + I (lanes 11 and 12) has a reduced TATA binding affinity, in agreement with previous reports (7Mittal V. Hernandez N. Science. 1997; 275: 1136-1140Crossref PubMed Scopus (77) Google Scholar, 8Henry R.W. Ford E. Mital R. Mittal V. Hernandez N. Cold Spring Harbor Symp. Quant. Biol. 1998; 63: 111-120Crossref PubMed Scopus (32) Google Scholar) and the addition of HMG-1 has no effect on stability (extended exposure in lanes 11′ and 12′ is shown in the right panel). To ensure that ΔN + I remained capable of binding to the TATA element and was not simply inactivated during the purification procedure, TFIIB was reacted with TBP/TATA to form a stable TFIIB/TBP/TATA complex (data not shown). There is no mobility shift and insignificant intensity change on reaction of HMG-1 with either ΔN + III or ΔN (lanes 7–10), indicating that HMG-1 has little or no interaction with them. The band intensity for the TBP/TATA complex with these latter two TBP mutants is greater than for the others, in agreement with previous findings that showed that the N terminus within hTBP reduces the binding affinity of TBP to the TATA element (7Mittal V. Hernandez N. Science. 1997; 275: 1136-1140Crossref PubMed Scopus (77) Google Scholar). The latter three deletion mutants, ΔN, ΔN + I, and ΔN + III, represent the second group, all of which exhibit no significant interaction with HMG-1. These findings indicate that the Q-tract in segment II is a major target for HMG-1 binding. If the Q-tract is important for the HMG-1 interaction, HMG-1 should compete with an antibody specific to the Q-tract region and reduce or eliminate the formation of a supershifted complex. Antibodies that are targeted to epitopes that are not directly involved in the HMG-1 binding should correspondingly yield a supershifted complex in the presence or absence of HMG-1. Fig. 7A shows the sequence for the first 95 residues in the N terminus of hTBP, highlighting the location of the epitopes for two monoclonal antibodies (mAb). mAb1C2 was originally reported to be specific for residues 53–62 (shown in parenthesis), which lies at the junction of segment I and II and within the Q-tract. Recently, it was shown to be specific for the Q-tract (37Trottier Y. Lutz Y. Stevanin G. Imbert G. Devys D. Cancel G. Saudou F. Weber C. David G. Tora L. Agid Y. Brice A. Mandel J. Nature. 1995; 378: 403-406Crossref PubMed Scopus (585) Google Scholar). On the other hand, mAb3G3 targets residues 1–10 in hTBP. In the first set of experiments, TBP, TATA, and the antibody (1C2 or 3G3) were incubated, followed by addition of increasing amounts of HMG-1. Lane 3 in Fig. 7 B shows that mAb1C2 produces a supershifted complex in the absence of HMG-1. The addition of increasing levels of HMG-1 produces the HMG-1/TBP/TATA complex as seen in the characteristic band for the complex (lanes 4–8). As seen in lane 5, the presence of HMG-1, at as low as 5 ng, competed effectively with mAb1C2 for binding to TBP and disrupted its binding to TBP/TATA, resulting in the loss of a supershifted complex. In contrast, the parallel experiments that used mAb3G3 (lanes 10–16) showed that HMG-1 binding did not disrupt antibody binding to TBP, as evident by the continued presence of the supershifted complex. These data indicate that HMG-1 and mAb1C2 compete for the same or overlapping sites and that the HMG-1 binding to TBP is stronger than that for mAb1C2 to TBP. On the other hand, HMG-1 and mAb3G3 do not compete for the same site(s) and bind simultaneously to different and non-overlapping sites. Fig. 7 C shows the titration in which the HMG-1/TBP/TATA complex was preestablished, and increasing levels of antibody were added in an attempt to compete with HMG-1 binding. The addition of increasing amounts of mAb1C2 to the complex did not displace HMG-1 from the complex. This antibod" @default.
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• W2044804986 date "2001-08-01" @default.
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• W2044804986 title "The Binding Interaction of HMG-1 with the TATA-binding Protein/TATA Complex" @default.
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• W2044804986 doi "https://doi.org/10.1074/jbc.m011792200" @default.
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