Mot1 Regulates the DNA Binding Activity of Free TATA-binding Protein in an ATP-dependent Manner
2003; Elsevier BV; Volume: 278; Issue: 15 Linguagem: Inglês
10.1074/jbc.m211445200
ISSN1083-351X
AutoresRussell P. Darst, Arindam Das-Gupta, Chunming Zhu, Jer-Yuan Hsu, Amy Vroom, Tamara Muldrow, David Auble,
Tópico(s)Bacterial Genetics and Biotechnology
ResumoMot1 is an essential Snf2/Swi2-relatedSaccharomyces cerevisiae protein that binds the TATA-binding protein (TBP) and removes TBP from DNA using ATP hydrolysis. Mot1 functions in vivo both as a repressor and as an activator of transcription. Mot1 catalysis of TBP·DNA disruption is consistent with its function as a repressor, but the Mot1 mechanism of activation is unknown. To better understand the physiologic role of Mot1 and its enzymatic mechanism, MOT1mutants were generated and tested for activity in vitro and in vivo. The results demonstrate a close correlation between the TBP·DNA disruption activity of Mot1 and its essentialin vivo function. Previous results demonstrated a large overlap in the gene sets controlled by Mot1 and NC2. Mot1 and NC2 can co-occupy TBP·DNA in vitro, and NC2 binding does not impair Mot1-catalyzed disruption of the complex. Residues on the DNA-binding surface of TBP are important for Mot1 binding and the Mot1·TBP binary complex binds very poorly to DNA and does not dissociate in the presence of ATP. However, the binary complex binds DNA well in the presence of the transition state analog ADP-AlF4. A model for Mot1 action is proposed in which ATP hydrolysis causes the Mot1 N terminus to displace the TATA box, leading to ejection of Mot1 and TBP from DNA. Mot1 is an essential Snf2/Swi2-relatedSaccharomyces cerevisiae protein that binds the TATA-binding protein (TBP) and removes TBP from DNA using ATP hydrolysis. Mot1 functions in vivo both as a repressor and as an activator of transcription. Mot1 catalysis of TBP·DNA disruption is consistent with its function as a repressor, but the Mot1 mechanism of activation is unknown. To better understand the physiologic role of Mot1 and its enzymatic mechanism, MOT1mutants were generated and tested for activity in vitro and in vivo. The results demonstrate a close correlation between the TBP·DNA disruption activity of Mot1 and its essentialin vivo function. Previous results demonstrated a large overlap in the gene sets controlled by Mot1 and NC2. Mot1 and NC2 can co-occupy TBP·DNA in vitro, and NC2 binding does not impair Mot1-catalyzed disruption of the complex. Residues on the DNA-binding surface of TBP are important for Mot1 binding and the Mot1·TBP binary complex binds very poorly to DNA and does not dissociate in the presence of ATP. However, the binary complex binds DNA well in the presence of the transition state analog ADP-AlF4. A model for Mot1 action is proposed in which ATP hydrolysis causes the Mot1 N terminus to displace the TATA box, leading to ejection of Mot1 and TBP from DNA. TATA-binding protein open reading frame temperature-sensitive glutathione S-transferase armadillo A critical step in the assembly of an active transcription complex at an RNA polymerase II promoter involves recruitment of TATA-binding protein (TBP)1 and TBP-associated factors (1Kotani T. Banno K. Ikura M. Hinnebusch A.G. Nakatani Y. Kawaichi M. Kokubo T. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 7178-7183Crossref PubMed Scopus (40) Google Scholar, 2Struhl K. Cell. 1999; 98: 1-4Abstract Full Text Full Text PDF PubMed Scopus (372) Google Scholar, 3Maldonado E. Hampsey M. Reinberg D. Cell. 1999; 99: 455-458Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). TBP recruitment and activity are influenced by a large number of transcription factors and components of the general transcription machinery, many of which can interact directly with TBP (3Maldonado E. Hampsey M. Reinberg D. Cell. 1999; 99: 455-458Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar, 4Pugh B.F. Gene (Amst.). 2000; 255: 1-14Crossref PubMed Scopus (156) Google Scholar, 5Berk A.J. Curr. Opin. Cell Biol. 1999; 11: 330-335Crossref PubMed Scopus (84) Google Scholar, 6Lee T.I. Young R.A. Genes Dev. 1998; 12: 1398-1408Crossref PubMed Scopus (158) Google Scholar). MOT1 was uncovered in genetic screens for factors that repress transcription driven by a weak promoter (7Davis J.L. Kunisawa R. Thorner J. Mol. Cell. Biol. 1992; 12: 1879-1892Crossref PubMed Scopus (150) Google Scholar, 8Jiang H. Xie Y. Houston P. Stemke-Hale K. Mortensen U.H. Rothstein R. Kodadek T. J. Biol. Chem. 1996; 271: 33181-33186Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar, 9Karnitz L. Morrison M. Young E.T. Genetics. 1992; 132: 351-359Crossref PubMed Google Scholar, 10Piatti S. Tazzi R. Pizzagalli A. Plevani P. Lucchini G. Chromosoma. 1992; 102: S107-S113Crossref PubMed Scopus (23) Google Scholar, 11Prelich G. Mol. Cell. Biol. 1997; 17: 2057-2065Crossref PubMed Google Scholar). Consistent with its function as a repressor, Mot1 was isolated independently as an ATP-dependent factor that disrupts the TBP·DNA complex (12Auble D.T. Hahn S. Genes Dev. 1993; 7: 844-856Crossref PubMed Scopus (137) Google Scholar). Mot1 binds the TBP·DNA complexin vitro (12Auble D.T. Hahn S. Genes Dev. 1993; 7: 844-856Crossref PubMed Scopus (137) Google Scholar) and contacts both TBP and about 17 bp of DNA upstream of the TATA box (13Darst R.P. Wang D. Auble D.T. EMBO J. 2001; 20: 2028-2040Crossref PubMed Scopus (36) Google Scholar). In the absence of DNA, Mot1 also dimerizes with TBP (13Darst R.P. Wang D. Auble D.T. EMBO J. 2001; 20: 2028-2040Crossref PubMed Scopus (36) Google Scholar, 14Poon D. Campbell A.M. Bai Y. Weil P.A. J. Biol. Chem. 1994; 269: 23135-23140Abstract Full Text PDF PubMed Google Scholar, 15Adamkewicz J.I. Mueller C.G. Hansen K.E. Prud'homme W.A. Thorner J. J. Biol. Chem. 2000; 275: 21158-21168Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). In this report, we refer to the Mot1·TBP complex as the “binary” complex, and the Mot1·TBP·DNA complex is referred to as the “ternary” complex. Mot1 homologs have been identified in many eukaryotes. The human homolog is BTAF1, which interacts with TBP (16van der Knaap J.A. Borst J.W. van der Vliet P.C. Gentz R. Timmers H.T. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 11827-11832Crossref PubMed Scopus (39) Google Scholar, 17Chicca 2nd, J.J. Auble D.T. Pugh B.F. Mol. Cell. Biol. 1998; 18: 1701-1710Crossref PubMed Scopus (59) Google Scholar) and catalyzes disruption of human TBP·DNA complexes (17Chicca 2nd, J.J. Auble D.T. Pugh B.F. Mol. Cell. Biol. 1998; 18: 1701-1710Crossref PubMed Scopus (59) Google Scholar). The insect homolog, the 89B helicase (18Goldman-Levi R. Miller C. Bogoch J. Zak N.B. Nucleic Acids Res. 1996; 24: 3121-3128Crossref PubMed Scopus (19) Google Scholar), may interact with TBP or TBP-related factor 1 (TRF1) in vitro (19Adamkewicz J.I. Hansen K.E. Prud'homme W.A. Davis J.L. Thorner J. J. Biol. Chem. 2001; 276: 11883-11894Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar). The Mot1 C terminus contains the conserved ATPase domain (7Davis J.L. Kunisawa R. Thorner J. Mol. Cell. Biol. 1992; 12: 1879-1892Crossref PubMed Scopus (150) Google Scholar), whereas the Mot1 N terminus is responsible for TBP binding (19Adamkewicz J.I. Hansen K.E. Prud'homme W.A. Davis J.L. Thorner J. J. Biol. Chem. 2001; 276: 11883-11894Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar, 20Auble D.T. Wang D. Post K.W. Hahn S. Mol. Cell. Biol. 1997; 17: 4842-4851Crossref PubMed Scopus (81) Google Scholar, 21Pereira L.A. van der Knaap J.A. van den Boom V. van den Heuvel F.A. Timmers H.T. Mol. Cell. Biol. 2001; 21: 7523-7534Crossref PubMed Scopus (28) Google Scholar). The structural basis for Mot1·TBP recognition is unknown, however, it was recently suggested that the Mot1 N terminus contains HEAT or ARM repeats, which compose a class of structurally related leucine-rich repeats (22Andrade M.A. Petosa C. O'Donoghue S.I. Muller C.W. Bork P. J. Mol. Biol. 2001; 309: 1-18Crossref PubMed Scopus (400) Google Scholar, 23Andrade M.A. Ponting C.P. Gibson T.J. Bork P. J. Mol. Biol. 2000; 298: 521-537Crossref PubMed Scopus (161) Google Scholar, 24Neuwald A.F. Hirano T. Genome Res. 2000; 10: 1445-1452Crossref PubMed Scopus (231) Google Scholar). Structural studies have shown that HEAT and ARM repeats form two α helices joined by a short loop (ARM repeats have a short additional α helix), and these can stack upon each other to form a “superhelix” that provides an extensive surface for macromolecular interaction (22Andrade M.A. Petosa C. O'Donoghue S.I. Muller C.W. Bork P. J. Mol. Biol. 2001; 309: 1-18Crossref PubMed Scopus (400) Google Scholar). Previous analysis of Mot1 deletion mutants indicated that an extended portion of the Mot1 N terminus is responsible for recognition of TBP (19Adamkewicz J.I. Hansen K.E. Prud'homme W.A. Davis J.L. Thorner J. J. Biol. Chem. 2001; 276: 11883-11894Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar, 20Auble D.T. Wang D. Post K.W. Hahn S. Mol. Cell. Biol. 1997; 17: 4842-4851Crossref PubMed Scopus (81) Google Scholar). It has also been reported that, in solution, Mot1 is a non-globular monomer (15Adamkewicz J.I. Mueller C.G. Hansen K.E. Prud'homme W.A. Thorner J. J. Biol. Chem. 2000; 275: 21158-21168Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). Taken together, these data suggest a model in which Mot1 adopts an extended conformation that provides a large surface for interaction with TBP. To test the model, mutations were made in both Mot1 and TBP, and the effects on the Mot1·TBP interaction were determined. Because HEAT and ARM repeats are based mostly on hydrophobic interactions (22Andrade M.A. Petosa C. O'Donoghue S.I. Muller C.W. Bork P. J. Mol. Biol. 2001; 309: 1-18Crossref PubMed Scopus (400) Google Scholar), it was expected that most polar residues in the N-terminal domain would not be essential, which we have found to be the case. Mot1 is a member of the Snf2/Swi2 ATPase family (25Gorbalenya A.E. Koonin E.V. Curr. Opin. Struct. Biol. 1993; 3: 419-429Crossref Scopus (1039) Google Scholar, 26Henikoff S. Trends Biochem. Sci. 1993; 18: 291-292Abstract Full Text PDF PubMed Scopus (40) Google Scholar, 27Eisen J.A. Sweder K.S. Hanawalt P.C. Nucleic Acids Res. 1995; 23: 2715-2723Crossref PubMed Scopus (619) Google Scholar). It has been suggested that at least some Snf2/Swi2 ATPases are processive molecular motors, acting by driving DNA translocation or rotation (28Havas K. Flaus A. Phelan M. Kingston R. Wade P.A. Lilley D.M. Owen-Hughes T. Cell. 2000; 103: 1133-1142Abstract Full Text Full Text PDF PubMed Scopus (227) Google Scholar, 29Cote J. Peterson C.L. Workman J.L. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 4947-4952Crossref PubMed Scopus (166) Google Scholar). The Mot1·TBP·DNA system has been used to test several theories about how these ATPases drive changes in protein·DNA interactions. Mot1 is not a helicase (13Darst R.P. Wang D. Auble D.T. EMBO J. 2001; 20: 2028-2040Crossref PubMed Scopus (36) Google Scholar, 14Poon D. Campbell A.M. Bai Y. Weil P.A. J. Biol. Chem. 1994; 269: 23135-23140Abstract Full Text PDF PubMed Google Scholar, 15Adamkewicz J.I. Mueller C.G. Hansen K.E. Prud'homme W.A. Thorner J. J. Biol. Chem. 2000; 275: 21158-21168Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar), nor does it travel long distances on DNA after TBP is removed from the TATA box (30Auble D.T. Steggerda S.M. Mol. Cell. Biol. 1999; 19: 412-423Crossref PubMed Scopus (25) Google Scholar). Catalysis of TBP·DNA disruption requires a grip by Mot1 on both upstream DNA and TBP, although the upstream DNA and the TBP·DNA complex can be conformationally uncoupled without impairing catalysis (13Darst R.P. Wang D. Auble D.T. EMBO J. 2001; 20: 2028-2040Crossref PubMed Scopus (36) Google Scholar). These results indicate that Mot1 does not dissociate TBP·DNA by propagation of DNA twist or writhe through the TATA box. A similar result has been reported for the Snf2/Swi2 family member ISWI (31Langst G. Becker P.B. Mol. Cell. 2001; 8: 1085-1092Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar). It is possible that Mot1 interacts with the TATA box directly and in so doing alters its structure or that Mot1 uses ATP hydrolysis to disrupt TBP·DNA complexes via short-range tracking or ATP-driven insertion of Mot1 into the TBP·DNA interface. Alternatively, Mot1 may mediate TBP·DNA disruption by inducing a conformational change in TBP that deforms the DNA-binding surface of TBP. Here we demonstrate that residues on the DNA-binding surface of TBP impair the interaction of TBP with Mot1, suggesting that Mot1 contacts the DNA-binding surface of TBP, and explaining why the Mot1·TBP binary complex binds DNA poorly compared with TBP alone. Binding of an ATP transition state analog locks the binary complex into a form in which the Mot1·TBP complex can bind DNA better than the nucleotide-free form of the Mot1·TBP complex. These results suggest that ATP hydrolysis causes a change in either the conformation of TBP or the interaction of Mot1 with the DNA-binding surface of TBP and that these ATP-driven conformational changes explain how Mot1 drives disruption of the TBP·DNA complex. Oligonucleotide primers flanking the EcoRI site (bp position 1026 in theMOT1 open reading frame (ORF)) and ClaI site (position 2092) were used to amplify ∼1 kb of the MOT1 ORF using Taq polymerase under reduced fidelity conditions as described previously (32Leung D.W. Chen E. Goeddel D.V. Technique. 1989; 1: 11-15Google Scholar). The PCR-amplified DNA was digested withEcoRI and ClaI and cloned into anEcoRI-ClaI-gapped plasmid containing the rest of the MOT1 ORF under control of the GAL1 promoter on a CEN ARS plasmid bearing the LEU2 gene (20Auble D.T. Wang D. Post K.W. Hahn S. Mol. Cell. Biol. 1997; 17: 4842-4851Crossref PubMed Scopus (81) Google Scholar). Note that an additional ClaI site is present in theMOT1 ORF, but this second site is blocked fromClaI digestion by overlapping dam methylation. Six independent transformants were picked at random from the bacterial transformation of the primary ligation mix, and these were sequenced and found to contain ∼1-bp change per kilobase (kb) of amplified DNA. Bacterial transformants containing the mutated DNA were then scrapeden masse from agar plates, inoculated at high density into liquid media, and used in a large-scale plasmid purification prep. The resulting purified plasmids were then used to transform yeast strain AY29 (mot1Δ::TRP1, carrying plasmid pMR13 (MOT1+ URA3+)) (20Auble D.T. Wang D. Post K.W. Hahn S. Mol. Cell. Biol. 1997; 17: 4842-4851Crossref PubMed Scopus (81) Google Scholar), which is otherwise congenic to YPH499 (33Sikorski R.S. Hieter P. Genetics. 1989; 122: 19-27Crossref PubMed Google Scholar) by selection on synthetic complete media containing glucose but without leucine using standard techniques (34Guthrie C. Fink G.R. Guide to Yeast Genetics and Molecular Biology. Academic Press, Inc., San Diego1991: 194Google Scholar). Approximately 13,000 transformants were replica-plated to synthetic glucose- or galactose-containing media lacking leucine and incubated at 30 °C for 3–5 days. Comparison of the glucose and galactose-containing plates did not reveal any GAL1-inducible alleles ofMOT1, which caused slow growth in the presence of wild-typeMOT1. Colonies were then replica-plated from galactose-containing media to media containing galactose and 5-fluoroorotic acid (34Guthrie C. Fink G.R. Guide to Yeast Genetics and Molecular Biology. Academic Press, Inc., San Diego1991: 194Google Scholar) to select for loss of theURA3-marked plasmid containing the wild-type MOT1gene. Approximately half of the transformants did not survive the 5-fluoroorotic acid selection, indicating that these strains harbored alleles of MOT1, which do not support growth in the absence of wild-type MOT1. The remaining viable strains were screened for temperature-sensitive growth defects by replica plating to synthetic galactose plates minus leucine and incubation at 30 °C and 35 °C. Temperature-sensitive strains were re-streaked, and the plasmids were isolated and re-transformed to the MOT1deletion strain to confirm the plasmid-linked temperature-sensitive (ts) phenotype. Candidate genes were then sequenced through the entire EcoRI-ClaI region of the ORF, and the mutant fragments were subcloned to a new plasmid backbone containing the remainder of the MOT1 gene to be sure that mutations in the EcoRI-ClaI DNA fragment were responsible for the phenotypes observed. Site-directed mutagenesis was performed using synthetic oligonucleotides and either overlapping PCR or the Stratagene QuikChange kit, according to the instructions provided by the manufacturer. Each mutation was engineered to encode a change in a restriction site (either introduction of a new site or loss of an existing site) to facilitate subcloning. Candidate transformants containing the correct restriction sites were then sequenced completely in a region that overlaps a DNA fragment with convenient restriction sites. The sequenced DNA fragment was then sub-cloned to LEU2 CEN ARS plasmids derived from pRS315 (33Sikorski R.S. Hieter P. Genetics. 1989; 122: 19-27Crossref PubMed Google Scholar) that contain theMOT1 ORF driven by the GAL1 promoter or by a 448-bp fragment of the MOT1 promoter. All constructs encode a Mot1 derivative with the Py tag (35Schneider K.R. Smith R.L. O'Shea E.K. Science. 1994; 266: 122-126Crossref PubMed Scopus (209) Google Scholar) appended to the N terminus to facilitate quantitation by Western blotting and purification using antibody-coupled beads (20Auble D.T. Wang D. Post K.W. Hahn S. Mol. Cell. Biol. 1997; 17: 4842-4851Crossref PubMed Scopus (81) Google Scholar, 35Schneider K.R. Smith R.L. O'Shea E.K. Science. 1994; 266: 122-126Crossref PubMed Scopus (209) Google Scholar). Additional details regarding plasmid construction are available upon request. Plasmids containing the site-directed alleles were transformed into AY29 yeast cells (see above), and the ability of the constructs to support viability was assessed by plasmid shuffling using standard techniques (34Guthrie C. Fink G.R. Guide to Yeast Genetics and Molecular Biology. Academic Press, Inc., San Diego1991: 194Google Scholar). Strains harboring alleles under control of the MOT1 promoter were analyzed for growth defects on synthetic media without leucine and containing raffinose, galactose, or glucose as the carbon source. Strains harboring alleles under control of the GAL1 promoter were streaked to galactose-containing plates to induce expression prior to plasmid shuffling. Growth of strains was compared with congenic wild-type cells by incubation at 16 °C, 30 °C, 32 °C, and 35 °C. Recombinant full-length TBP and TBP mutants expressed under the control of the T7 promoter as a fusion with N-terminal six-histidine tag were obtained by transformation of BL21(DE3) Escherichia coli cells with the appropriate plasmid expression vectors (13Darst R.P. Wang D. Auble D.T. EMBO J. 2001; 20: 2028-2040Crossref PubMed Scopus (36) Google Scholar, 36Kim T.K. Roeder R.G. J. Biol. Chem. 1997; 272: 7540-7545Abstract Full Text Full Text PDF PubMed Scopus (4) Google Scholar). Cells were inoculated into 1 liter of yeast extract Tryptone (YT) media containing 100 μg/ml ampicillin or 30 μg/ml kanamycin at 37 °C and were grown to an optical density at 600 nm of 0.7–1.0. Isopropyl-β-d-thiogalactopyranoside was added (0.5 mm final concentration), and the cells were incubated at 37 °C for 3 h to allow protein expression. Cells were harvested and resuspended in buffer I (40 mm sodium phosphate, pH 8.0, 300 mm NaCl, 10% glycerol, 1 mmphenylmethylsulfonyl fluoride, 2 μm benzamidine, 2 μm pepstatin, 0.6 μm leupeptin, and 2 μg/ml chymostatin) containing 5 mm imidazole and lysed by sonication. After sonication, the cleared lysate was incubated at 4 °C for 1 h with 0.2 ml of nickel-nitrilotriacetic acid-agarose (Qiagen) pre-equilibrated with buffer I, which included 5 mm imidazole. The mixture was then loaded into a column, and the resin was washed with 10 ml buffer I plus 5 mmimidazole and subsequently with 5 ml of buffer I containing 20 mm imidazole. Finally, the bound protein was eluted with buffer I containing 200 mm imidazole. The yield was quantitated by Bradford assay (Bio-Rad), and the purity was assessed by Coomassie Blue staining of 10% protein gels. Based on the Coomassie Blue staining, the proteins were estimated to be about 90% pure. For detection of TBP by native gel electrophoresis (Figs. 6C, 6D, and7B), full-length TBP and Mot1 were incubated in binding buffer (13Darst R.P. Wang D. Auble D.T. EMBO J. 2001; 20: 2028-2040Crossref PubMed Scopus (36) Google Scholar) containing 120 mm KCl and 12 mmHEPES buffer, pH 7.6 (37Banik U. Beechem J.M. Klebanow E. Schroeder S. Weil P.A. J. Biol. Chem. 2001; 276: 49100-49109Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar), using proteins at the concentrations indicated in figure legends. The gels in Figs. 6C and7B were run with the electrodes reversed: samples were loaded on the side of the positive electrode, and run toward the negative electrode. The gel was, however, pre-run for >50 min with the electrodes connected in the usual fashion before loading. Following electrophoresis, the gels were boiled in 1% SDS for 1 min, then transferred to Immobilon and TBP was detected using TBP antiserum. The TBE gel shift assay in Fig. 8A was performed as described previously (38Cang Y. Auble D.T. Prelich G. EMBO J. 1999; 18: 6662-6671Crossref PubMed Scopus (55) Google Scholar). Gel shift assays were otherwise performed as described previously (13Darst R.P. Wang D. Auble D.T. EMBO J. 2001; 20: 2028-2040Crossref PubMed Scopus (36) Google Scholar) using 5 nm core domain TBP (gift of J. Geiger) or full-length TBP with minor modifications as indicated. Synthesis and labeling of the 36- and 17-bp DNAs, and preparation of the radiolabeled 100-bp adenovirus major late promoter fragment, was as previously described (13Darst R.P. Wang D. Auble D.T. EMBO J. 2001; 20: 2028-2040Crossref PubMed Scopus (36) Google Scholar). DNA concentration was about 0.5 nm in the reactions. The concentration of Mot1 needed to bind 50% of the TBP·DNA complex is ∼5 nm (13Darst R.P. Wang D. Auble D.T. EMBO J. 2001; 20: 2028-2040Crossref PubMed Scopus (36) Google Scholar). The concentration of Mot1 used was estimated from this activity and is indicated in the figure legends. ATP was used at between 5 and 100 μm. ADP was used at 100 μm. NaF was used at 2.5 mm. AlCl3 was used at 10 μm(39Maruta S. Henry G.D. Sykes B.D. Ikebe M. J. Biol. Chem. 1993; 268: 7093-7100Abstract Full Text PDF PubMed Google Scholar). Bur6 was used at 13 nm and Ydr1/Ncb2 at 60 nm (38Cang Y. Auble D.T. Prelich G. EMBO J. 1999; 18: 6662-6671Crossref PubMed Scopus (55) Google Scholar); both proteins were a gift of G. Prelich.Figure 7ATP transition state analog facilitates binding of Mot1·TBP complex to DNA. A, gel mobility shift assay using radiolabeled 0.5 nm TATA DNA and 5 nm TBP. Approximately 40 nm Mot1, 5 μm ATP, and ADP-AlF4 (see “Materials and Methods”) were added where indicated. Orders of addition of DNA, TBP, Mot1, ATP, and ADP-AlF4 are indicated. In the first incubation (“Added 1st”), components were incubated for 30 min. Additional components were then added as indicated (“Added 2nd”), and the reactions were incubated for 30 min and loaded onto the gel. ATP was added 10 min after AlCl3 in lane 13. The position of the Mot1·TBP·DNA ternary complex is marked “3o.” Phosphorimaging analysis showed that the ternary complex band inlane 6 obtained after preincubation of Mot1 and TBP is only 10% the intensity of the ternary complex band in lane 3. The ternary complex bands in lanes 5 and 8 are both 50% the intensity of the ternary complex band in lane 3, indicating 50% loss of the ternary complex in the presence of ADP-AlF4, but that ADP-AlF4 completely restores the ability of pre-formed Mot1·TBP complexes to bind to DNA.B, Mot1 alone (lane 1), TBP alone (lanes 2, 3, and 7) or TBP plus Mot1 (lanes 4–6) were incubated in the absence (lanes 1,4, and 7) or presence of ATP (lanes 3and 6) or ADP-AlF4 (lanes 2 and 5). Proteins were incubated with ATP or ADP-AlF4for a total of 30 min. In reactions with both Mot1 and TBP, Mot1 was incubated with ATP or ADP-AlF4 for 20 min, followed by addition of TBP for 10 min. The reactions were loaded onto non-denaturing polyacrylamide gels and electrophoresed as in Fig.6C. Following electrophoresis, Western analysis was performed to detect TBP. The position of TBP is shown. Mot1 and TBP were used at 50 nm, ATP was 100 μm, and ADP-AlF4 was used as described under “Materials and Methods.”View Large Image Figure ViewerDownload (PPT)Figure 8Mot1 binds and disrupts the TBP·NC2·DNA complex. A, gel mobility shift analysis using a Tris borate-EDTA (TBE) native gel, 0.5 nmradiolabeled TATA DNA, and 5 nm TBP. The positions of the various complexes are indicated by the arrows. Mot1 (5 nm) and NC2 (composed of Bur6, 13 nm, and Ydr1/Ncb2, 60 nm) were added where indicated. B, gel mobility shift analysis using a Tris-glycine (TG) native gel was performed using radiolabeled TATA DNA, TBP, Mot1, and NC2 as inA. The bracket in lane 3 marks the Mot1·TBP·DNA shift, which was not discrete in this experiment. Theasterisk indicates the distinct Mot1·NC2·TBP·DNA shift.View Large Image Figure ViewerDownload (PPT) Mot1 was expressed and purified from yeast using antibody-coupled beads exactly as described previously (20Auble D.T. Wang D. Post K.W. Hahn S. Mol. Cell. Biol. 1997; 17: 4842-4851Crossref PubMed Scopus (81) Google Scholar). The antibody-coupled beads were prepared using Py monoclonal antibody that recognizes the Mot1 epitope tag (20Auble D.T. Wang D. Post K.W. Hahn S. Mol. Cell. Biol. 1997; 17: 4842-4851Crossref PubMed Scopus (81) Google Scholar), which was prepared at the University of Virginia Lymphocyte Culture Center. For detection of TBP binding to immobilized Mot1, Mot1-coupled beads were equilibrated with buffer T-60 (30 mm Tris (pH 8.0), 5 mm magnesium chloride, 0.1% Brij-58, 1 mm dithiothreitol, protease inhibitors plus 60 mm potassium chloride). One hundred nanograms of full-length recombinant yeast TBP (or TBP mutant) was added in 500 μl of buffer T-60, and the reaction was incubated for 30 min at room temperature. After binding, the unbound material was collected and the beads were washed with buffer T containing increasing concentrations of KCl; samples marked “Eluate” were collected in T-1000. The eluted proteins were precipitated with acetone, and TBP present in the eluates was detected by Western blotting using rabbit polyclonal anti-TBP antisera. One-liter cultures of DH10B bacterial cells containing plasmid pGEX-1 (Amersham Biosciences) or a plasmid expressing GST fused to full-length yeast TBP (kindly provided by Ron Reeder) were grown in YT medium at 37 °C to an optical density at 600 nm of 0.7–1.0. Isopropyl-β-d-thiogalactopyranoside was added (1.0 mm, final concentration), and the cells were incubated at 37 °C for an additional 3 h. Cells were harvested by centrifugation and resuspended in 20 ml of buffer T (30 mmTris-HCl, pH 8.0, 2 μm pepstatin A, 1 mmphenylmethylsulfonyl fluoride) containing 150 mm KCl (T-150 buffer). Cells were lysed by sonication, and debris was removed by centrifugation. After centrifugation, 0.5 ml of GST lysate or 1.5 ml of GST-TBP lysate was incubated at 4 °C for 1 h with 20 μl of glutathione-agarose equilibrated with buffer T containing 60 mm KCl (T-60). The agarose was washed three times with 1 ml of T-150 buffer, and the entire 20-μl sample of agarose-bound material was used for testing the binding of Mot1. 10 ng of the eluted Mot1 protein obtained from yeast overexpression strains (see above) was added to a 20-μl suspension of GST or GST-TBP agarose in T-60 buffer and incubated on a roller for 1 h at 4 °C. The agarose was washed once with 0.6 ml of T-60 buffer, then an elution step was carried out with 0.6 ml of T-60 buffer with 5 mmMgCl2, with or without 50 μm ATP and with or without 1 nm TATA sequence DNA. Eluted proteins were precipitated with acetone for analysis by Western blotting using the Py antibody (35Schneider K.R. Smith R.L. O'Shea E.K. Science. 1994; 266: 122-126Crossref PubMed Scopus (209) Google Scholar), which recognizes the N-terminal epitope tag. Blocks of conserved sequences in the Mot1 N terminus were identified with a set of Mot1 homologs found in Entrez protein sequence data bank (available at www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=Protein) (from Homo sapiens (accession number AAC04573), Arabidopsis thaliana (T47857), Saccharomyces cerevisiae (P32333),Schizosaccharomyces pombe (T40642), and Drosophila melanogaster (AAF55260)) using the MACAW (40Schuler G.D. Altschul S.F. Lipman D.J. Proteins Struct. Funct. Genet. 1991; 9: 180-190Crossref PubMed Scopus (895) Google Scholar) and ClustalW algorithms (available at www.ibc.wustl.edu/service/msa/index.html). HEAT repeats in Mot1 reported by previous authors (23Andrade M.A. Ponting C.P. Gibson T.J. Bork P. J. Mol. Biol. 2000; 298: 521-537Crossref PubMed Scopus (161) Google Scholar, 24Neuwald A.F. Hirano T. Genome Res. 2000; 10: 1445-1452Crossref PubMed Scopus (231) Google Scholar) included five in the B block. We observed sequence similarity of the second B block HEAT repeat to sequences immediately downstream using MACAW. This had not been found in the original sequence analysis, and Fig.1B therefore includes an additional HEAT repeat in the B block, for a total of six. Alignment of Mot1 homologs revealed conserved blocks outside of the ATPase, which we designate A–D (Fig. 1A). These blocks were not found in any protein except Mot1 or its homologs. The A and B blocks in the human and yeast proteins are about 40% identical. For example, Fig. 1B shows the sequence of theS. cerevisiae Mot1 A block; the asterisksindicate residues th
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