High Affinity Interaction of Yeast Transcriptional Regulator, Mot1, with TATA Box-binding Protein (TBP)
2001; Elsevier BV; Volume: 276; Issue: 15 Linguagem: Inglês
10.1074/jbc.m010665200
ISSN1083-351X
AutoresJoanne I. Adamkewicz, Karin E. Hansen, Wendy A. Prud'homme, Jennifer L. Davis, Jeremy Thorner,
Tópico(s)DNA Repair Mechanisms
ResumoYeast Mot1, an essential ATP-dependent regulator of basal transcription, removes TATA box-binding protein (TBP) from TATA sites in vitro. Complexes of Mot1 and Spt15 (yeast TBP), radiolabeled in vitro, were immunoprecipitated with anti-TBP (or anti-Mot1) antibodies in the absence of DNA, showing Mot1 binds TBP in solution. Mot1 N-terminal deletions (residues 25–801) abolished TBP binding, whereas C-terminal ATPase domain deletions (residues 802–1867) did not. Complex formation was prevented above 200 mm salt, consistent with electrostatic interaction. Correspondingly, TBP variants lacking solvent-exposed positive charge did not bind Mot1, whereas a mutant lacking positive charge within the DNA-binding groove bound Mot1. ATPase-defective mutant, Mot1(D1408N), which inhibits growth when overexpressed (but is suppressed by co-overexpression of TBP), bound TBP normally in vitro, suggesting it forms nonrecyclable complexes. N-terminal deletions of Mot1(D1408N) were not growth-inhibitory. C-terminal deletions were toxic when overexpressed, and toxicity was ameliorated by TBP co-overproduction. Residues 1–800 of Mot1 are therefore necessary and sufficient for TBP binding. The N terminus of 89B, a tissue-specific Drosophila Mot1 homolog, bound the TBP-like factor, dTRF1. Native Mot1 and derivatives deleterious to growth localized in the nucleus, whereas nontoxic derivatives localized to the cytosol, suggesting TBP binding and nuclear transport of Mot1 are coupled. Yeast Mot1, an essential ATP-dependent regulator of basal transcription, removes TATA box-binding protein (TBP) from TATA sites in vitro. Complexes of Mot1 and Spt15 (yeast TBP), radiolabeled in vitro, were immunoprecipitated with anti-TBP (or anti-Mot1) antibodies in the absence of DNA, showing Mot1 binds TBP in solution. Mot1 N-terminal deletions (residues 25–801) abolished TBP binding, whereas C-terminal ATPase domain deletions (residues 802–1867) did not. Complex formation was prevented above 200 mm salt, consistent with electrostatic interaction. Correspondingly, TBP variants lacking solvent-exposed positive charge did not bind Mot1, whereas a mutant lacking positive charge within the DNA-binding groove bound Mot1. ATPase-defective mutant, Mot1(D1408N), which inhibits growth when overexpressed (but is suppressed by co-overexpression of TBP), bound TBP normally in vitro, suggesting it forms nonrecyclable complexes. N-terminal deletions of Mot1(D1408N) were not growth-inhibitory. C-terminal deletions were toxic when overexpressed, and toxicity was ameliorated by TBP co-overproduction. Residues 1–800 of Mot1 are therefore necessary and sufficient for TBP binding. The N terminus of 89B, a tissue-specific Drosophila Mot1 homolog, bound the TBP-like factor, dTRF1. Native Mot1 and derivatives deleterious to growth localized in the nucleus, whereas nontoxic derivatives localized to the cytosol, suggesting TBP binding and nuclear transport of Mot1 are coupled. In yeast (Saccharomyces cerevisiae), as in other eukaryotes, many positive and negative regulatory factors act in concert to ensure that transcription initiation by RNA polymerase II (pol II)1 is controlled correctly (1Hampsey M. Microbiol. Mol. Biol. Rev. 1998; 62: 465-503Crossref PubMed Google Scholar). Promoter accessibility is dictated by chromatin structure, which is modulated by chromatin-remodeling complexes and histone-modifying enzymes (2Kornberg R.D. Lorch Y. Curr. Opin. Genet. & Dev. 1999; 9: 148-151Crossref PubMed Scopus (202) Google Scholar). Once a promoter is available, formation of a preinitiation complex begins with binding of TFIID, a complex composed of the TATA box-binding protein (TBP) and several TBP-associated factors (TAFs) (3Albright S.R. Tjian R. Gene ( Amst. ). 2000; 242: 1-13Crossref PubMed Scopus (275) Google Scholar). TFIID binds to the TATA box sequence present at nearly all yeast promoters (4Struhl K. Annu. Rev. Genet. 1995; 29: 651-674Crossref PubMed Scopus (136) Google Scholar, 5Kornberg R.D. Trends Cell Biol. 1999; 9: M46-M49Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar). Formation of a TFIID-DNA complex creates a platform for assembly of the remaining general transcription factors and pol II (6Roeder R.G. Trends Biochem. Sci. 1996; 21: 327-335Abstract Full Text PDF PubMed Scopus (718) Google Scholar, 7Travers A. Curr. Biol. 1996; 6: 401-403Abstract Full Text Full Text PDF PubMed Scopus (4) Google Scholar, 8Myer V.E. Young R.A. J. Biol. Chem. 1998; 273: 27757-27760Abstract Full Text Full Text PDF PubMed Scopus (171) Google Scholar). Transcriptional activators stimulate transcription initiation via recruitment of the pol II preinitiation complex to a promoter either through direct interaction with TBP, TFIIB, and/or components of the pol II holoenzyme (9Stargell L.A. Struhl K. Trends Genet. 1996; 12: 311-315Abstract Full Text PDF PubMed Scopus (103) Google Scholar, 10Parvin J.D. Young R.A. Curr. Opin. Genet. & Dev. 1998; 8: 565-570Crossref PubMed Scopus (49) Google Scholar) or via intervening “mediator” complexes (11Malik S. Roeder R.G. Trends Biochem. Sci. 2000; 25: 277-283Abstract Full Text Full Text PDF PubMed Scopus (305) Google Scholar). Considerable evidence highlights the importance of activator recruitment of TBP. For example, by using a TBP derivative with altered DNA binding specificity, it was shown that activator-dependent engagement of TBP at a promoter is rate-limiting for transcription in vivo (12Klein C. Struhl K. Science. 1994; 266: 280-282Crossref PubMed Scopus (106) Google Scholar). Also, fusion of TBP to the DNA-binding domains of either LexA or Gal4 allows high level transcription from LexA- or Gal4-binding sites in the absence of any other activator (13Chatterjee S. Struhl K. Nature. 1995; 374: 820-822Crossref PubMed Scopus (167) Google Scholar, 14Klages N. Strubin M. Nature. 1995; 374: 822-823Crossref PubMed Scopus (154) Google Scholar, 15Xiao H. Friesen J.D. Lis J.T. Mol. Cell. Biol. 1995; 15: 5757-5761Crossref PubMed Scopus (102) Google Scholar). Finally, as shown by in vivocross-linking techniques, promoter occupancy by TBP correlates with transcriptional activity (16Kuras L. Struhl K. Nature. 1999; 399: 609-613Crossref PubMed Scopus (399) Google Scholar, 17Li X.Y. Virbasius A. Zhu X. Green M.R. Nature. 1999; 399: 605-609Crossref PubMed Scopus (206) Google Scholar). Because promoter occupancy by TBP is central to gene activation (18Pugh B.F. Gene ( Amst. ). 2000; 255: 1-14Crossref PubMed Scopus (156) Google Scholar), TBP is also a prime target of transcriptional inhibitors. A conserved heterodimeric repressor, NC2/Dr1/DRAP, binds TBP via a histone-like fold, preventing TBP association with TFIIA and/or TFIIB, thereby repressing transcription (19Inostroza J.A. Mermelstein F.H. Ha I. Lane W.S. Reinberg D. Cell. 1992; 70: 477-489Abstract Full Text PDF PubMed Scopus (293) Google Scholar, 20Kim T.K. Zhao Y. Ge H. Bernstein R. Roeder R.G. J. Biol. Chem. 1995; 270: 10976-10981Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar, 21Goppelt A. Stelzer G. Lottspeich F. Meisterernst M. EMBO J. 1996; 15: 3105-3116Crossref PubMed Scopus (131) Google Scholar, 22Mermelstein F. Yeung K. Cao J. Inostroza J.A. Erdjument-Bromage H. Eagelson K. Landsman D. Levitt P. Tempst P. Reinberg D. Genes Dev. 1996; 10: 1033-1048Crossref PubMed Scopus (113) Google Scholar). S. cerevisiae cells lacking NC2α (BUR6/NCB1 gene product) grow very poorly; cells lacking NC2β (NCB2 gene product) are inviable (21Goppelt A. Stelzer G. Lottspeich F. Meisterernst M. EMBO J. 1996; 15: 3105-3116Crossref PubMed Scopus (131) Google Scholar, 23Prelich G. Mol. Cell. Biol. 1997; 17: 2057-2065Crossref PubMed Google Scholar). Another class of negative transcriptional regulator targeting TBP is the yeast MOT1 gene product. Like NC2, Mot1 is essential for viability and evolutionarily conserved. Mot1 homologs exist inDrosophila (55Goldman-Levi R. Miller C. Bogoch J. Zak N.B. Nucleic Acids Res. 1996; 24: 3121-3128Crossref PubMed Scopus (19) Google Scholar) and humans (63van 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, 64Chicca J.J., II Auble D.T. Pugh B.F. Mol. Cell. Biol. 1998; 18: 1701-1710Crossref PubMed Scopus (59) Google Scholar). A recessive temperature-sensitive mutation (mot1-1) elevated transcription of a plasmid-borne reporter gene in the absence of its normal activator and also increased transcription from several chromosomal pol II-dependent genes, as well as from a basal promoter lacking any upstream activation sequence (UAS) (24Davis J.L. Kunisawa R. Thorner J. Mol. Cell. Biol. 1992; 12: 1879-1892Crossref PubMed Scopus (150) Google Scholar). An independently isolated allele (mot1-1033) had a similar effect on another pol II gene (25Piatti S. Tazzi R. Pizzagalli A. Plevani P. Lucchini G. Chromosoma. 1992; 102: S107-S113Crossref PubMed Scopus (23) Google Scholar). Another recessive allele (bur3-1, for “bypass UAS requirement,” nowmot1-301) was isolated by selecting for mutations permitting transcription from the SUC2 gene promoter lacking its UAS (23Prelich G. Mol. Cell. Biol. 1997; 17: 2057-2065Crossref PubMed Google Scholar, 26Prelich G. Winston F. Genetics. 1993; 135: 665-676Crossref PubMed Google Scholar). Consistent with a primary role as a negative regulator, Mot1 was found to be the factor responsible for anATP-dependent inhibitory activity (ADI) in nuclear extracts (27Auble D.T. Hahn S. Genes Dev. 1993; 7: 844-856Crossref PubMed Scopus (137) Google Scholar) that removes TATA box-bound TBP in an ATP-dependent manner (28Auble D.T. Hansen K.E. Mueller C.G. Lane W.S. Thorner J. Hahn S. Genes Dev. 1994; 8: 1920-1934Crossref PubMed Scopus (277) Google Scholar). Although TBP stimulates RNA synthesis by all three RNA polymerase types in transcription reactionsin vitro (29Cormack B.P. Struhl K. Cell. 1992; 69: 685-696Abstract Full Text PDF PubMed Scopus (277) Google Scholar), the presence of Mot1 decreases transcription of pol II genes, but not pol I or pol III genes (28Auble D.T. Hansen K.E. Mueller C.G. Lane W.S. Thorner J. Hahn S. Genes Dev. 1994; 8: 1920-1934Crossref PubMed Scopus (277) Google Scholar). Likewise, Mot1 is required for repression in vitro of LEU2expression by the negative regulator, Leu3 (30Wade P.A. Jaehning J.A. Mol. Cell. Biol. 1996; 16: 1641-1648Crossref PubMed Scopus (35) Google Scholar). Mot1 was also identified in nuclear fractionation studies as a component (Taf170) of TBP-containing complexes distinct from TFIID (31Poon D. Campbell A.M. Bai Y. Weil P.A. J. Biol. Chem. 1994; 269: 23135-23140Abstract Full Text PDF PubMed Google Scholar). The C-terminal domain of Mot1 possesses the seven signature motifs of a superfamily of helicases and nucleic acid-dependent ATPases (32Gorbalenya E.G. Koonin E.V. Donchenko A.P. Blinov V.M. Nucleic Acids Res. 1989; 17: 4713-4730Crossref PubMed Scopus (830) Google Scholar), many of which modulate the state of assembly of protein-nucleic acid complexes (33Pazin M.J. Kadonaga J.T. Cell. 1997; 88: 737-740Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar). Mot1 is the prototype of a distinct class that includes Snf2 (yeast), ERCC6/CSB (human), Brahma (Drosophila), HepA (Escherichia coli), and others (24Davis J.L. Kunisawa R. Thorner J. Mol. Cell. Biol. 1992; 12: 1879-1892Crossref PubMed Scopus (150) Google Scholar, 34Eisen J.A. Sweder K.S. Hanawalt P.C. Nucleic Acids Res. 1995; 23: 2715-2723Crossref PubMed Scopus (619) Google Scholar). Despite this homology, no helicase activity has been reported for any member of the Mot1/Snf2 group. Mot1, purified to apparent homogeneity, does not exhibit even local DNA strand-unwinding activity but does retain full ability to release TBP bound to a TATA box in an ATP-dependent manner (35Adamkewicz J.I. Mueller C.G. Hansen K.H. Prud'homme W. Thorner J. J. Biol. Chem. 2000; 275: 21158-21168Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar). Prior studies (36Auble D.T. Wang D. Post K.W. Hahn S. Mol. Cell. Biol. 1997; 17: 4842-4851Crossref PubMed Scopus (81) Google Scholar) suggested that contacts with DNA do not play a critical role in Mot1-TBP interaction. Here we have explored this issue in detail and present experiments performed both in vivo and in vitro that define the requirements for, and quantify the affinity of, the physical association between Mot1 and TBP. We also determined the subcellular localization of Mot1, and the role of TBP interaction in Mot1 compartmentation. Strain W303-1A (MAT a ura3-1 his3-11,15 ade2-1 leu2-3,112 trp1-1 can1-100) was used for all experiments, unless otherwise indicated. For immunofluorescence, an otherwise isogenic MAT a/MATα diploid (W303D) was used. Strain KHY18 was constructed in the following manner. Strain YPH501 (37Sikorski R.S. Hieter P. Genetics. 1989; 122: 19-27Crossref PubMed Google Scholar) was transformed (38Gietz D. St. Jean A. Woods R.A. Schiestl R.H. Nucleic Acids Res. 1992; 20: 1425Crossref PubMed Scopus (2899) Google Scholar) with anXhoI-NotI fragment of plasmid pKH4 containing themot1-Δ2::LEU2 allele in which theLEU2 gene replaces codons 79–1822 of theMOT1-coding sequence (39Hansen K. Biochemical and Genetic Characterization of Mot1 and Its Interaction with Components of the Basal Transcription Complex in Saccharomyces cerevisiae Ph.D. thesis. University of California, Berkeley1996Google Scholar). After confirming correct transplacement of the MOT1 locus on one homolog of chromosome XVI by Southern blotting, the resultingLEU2 + diploid (KHY1) was transformed with pRSMOT1, a derivative of the URA3-markedCEN-based vector, pRS316 (37Sikorski R.S. Hieter P. Genetics. 1989; 122: 19-27Crossref PubMed Google Scholar), containing a 7.1-kb genomicBglII fragment expressing MOT1 from its endogenous promoter (24Davis J.L. Kunisawa R. Thorner J. Mol. Cell. Biol. 1992; 12: 1879-1892Crossref PubMed Scopus (150) Google Scholar). After sporulation and tetrad dissection, one of the resulting URA3 + LEU2 + MAT a spore clones was designated KHY18 and was used to study two site-directed mutants (mot1-462 and mot1-503) generated in this study (see below). Strain WPY1 was generated by transformation of strain W303D with a BamHI-SnaBI fragment of plasmid pKA23 containing the spt15Δ::LEU2mutation, in which a portion of the SPT15 coding sequence has been replaced with the LEU2 gene (40Arndt K.M. Ricupero S.L. Eisenmann D.M. Winston F. Mol. Cell. Biol. 1992; 12: 2372-2382Crossref PubMed Scopus (32) Google Scholar). After selection of LEU2 + transformants and verification for proper integration at the SPT15 locus using the polymerase chain reaction (PCR) with appropriate primers, WPY1 was transformed with a TRP1-marked CEN-based plasmid expressing wild-type SPT15 from its endogenous promoter (pUN45IID) or the same plasmid expressing either of two TBP mutants (K133L,K138L or K133L,K145L) (41Buratowski S. Zhou H. Science. 1992; 255: 1130-1132Crossref PubMed Scopus (82) Google Scholar). After sporulation, the cells were treated with diethyl ether to enrich for spores (42Dawes I.W. Hardie I.D. Mol. Gen. Genet. 1974; 131: 281-289Crossref PubMed Scopus (76) Google Scholar), andLEU2 + TRP1 + haploids carrying the spt15Δ::LEU2 mutation, maintained by low copy plasmids expressing TBP or TBP mutants, were selected on medium lacking Leu and Trp. Yeast cells without plasmids were grown at 30 °C in rich medium (YP), and cells with plasmids were grown in minimal medium (SC) lacking the appropriate nutrient(s) to maintain selection, as described (43Sherman F. Fink G.R. Hicks J.B. Methods in Yeast Genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1986Google Scholar), using glucose (Glc) as the carbon source unless otherwise indicated. Solid media contained either 2% Glc or 2% galactose and 0.2% sucrose (Gal/Suc). For expression of genes from the galactose-inducibleGAL1 promoter in liquid media, cells were pre-grown in SC containing 2% raffinose (Raf) as the carbon source, and then galactose (Gal) was added to 2% to initiate induction. Plasmids were introduced into yeast cells using a modification of the lithium acetate transformation procedure that utilizes single-stranded carrier DNA (38Gietz D. St. Jean A. Woods R.A. Schiestl R.H. Nucleic Acids Res. 1992; 20: 1425Crossref PubMed Scopus (2899) Google Scholar). Recombinant DNA manipulations were carried out using standard techniques (44Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). To facilitate immunodetection of Mot1, DNA encoding the 16-residue c-Myc epitope (LEEQKLISEEDLLRKR) recognized by the monoclonal antibody (mAb) 9E10 (45Evan G.I. Lewis G.K. Ramsay G. Bishop M.J. Mol. Cell. Biol. 1985; 5: 3610-3616Crossref PubMed Scopus (2166) Google Scholar) was fused in-frame to the 3′-end of the MOT1coding sequence, using a PCR-based method that exploits 3 primers (46Yon J. Fried M. Nucleic Acids Res. 1989; 17: 4895Crossref PubMed Scopus (241) Google Scholar) as follows: an upstream primer (JDM26), aMOT1/Myc joiner primer (JDM25), and a Myc epitope-containing primer (MYC) (TableI). The resulting 674-base pair PCR product was introduced into theSmaI site of plasmid pRS316 (37Sikorski R.S. Hieter P. Genetics. 1989; 122: 19-27Crossref PubMed Google Scholar) by blunt-end ligation and verified by DNA sequencing. The resulting product was digested withBstXI and introduced into BstXI-digested pRSMOT1, yielding pRSMot1Myc. Addition of the Myc tag did not detectably alter Mot1 function since pRSMot1 fully complements amot1Δ mutation (data not shown). A Myc-tagged version of the dominant-negative ATPase-defectiveMOT1(D1408N) allele (28Auble D.T. Hansen K.E. Mueller C.G. Lane W.S. Thorner J. Hahn S. Genes Dev. 1994; 8: 1920-1934Crossref PubMed Scopus (277) Google Scholar) was prepared in the same fashion, yielding pRSMot1(D1408N)Myc.Table ISynthetic oligonucleotide primers used for PCRNameSequence (5′ to 3′)JDM6AACCTCACTAGAACGTAGJDM25AAGTAGTTTTGAAATGCTGAGCTCCTCGTCJDM26GACATGGTTGAAAATGKHP5CGCTACGTTCTAGTGAGGKHP6GCCGCTCGAGTGGCTTAGCTTTAGAAGGATCKHP9CCGCTCGAGACCATGGCGGAAGAGGCTCAACTAAKHP12AATTAACCCTCACTAAAGGGAGCATGGCAATCAATTCAGCCAAAGKHP13AATTAACCCTCACTAAAGGGAGCATGGATCCTTCTAAAGCTAAGCKHP14AATGACCTTGTATACGCGTCKHP15AATTAACCCTCACTAAAGGGAGCATGGAATCACAATATATTCTAAAGCCKHP16AATTAACCCTCACTAAAGGGAGCATGGATCTATTGGCAAAGTTGTGCGKHP22AATTAACCCTCACTAAAGGGAACCATGGCGTCACGAGTTTCGAGGCTGGKHP23AATTAACCCTCACTAAAGGGAACCATGGTGAAGTTGGAACATGAAATGAA AATAMYCTTACCTCTTCCTGAGGAGGTCCTCTTCGCTGATTAATTTCTGCTCCTCGAGT7GTAATACGACTCACTATAGGGCG Open table in a new tab A series of truncations and internal deletions of MOT1Mycand MOT1(D1408N)Myc (see Fig. 4) expressed from the GAL1 promoter and carried onURA3-containing 2-μm DNA-based plasmids were generated, as appropriate, from plasmids, pKH7 or pKH14, respectively (28Auble D.T. Hansen K.E. Mueller C.G. Lane W.S. Thorner J. Hahn S. Genes Dev. 1994; 8: 1920-1934Crossref PubMed Scopus (277) Google Scholar), as described below. To produce pKH23, pKH33, pKH34, pKH35, pKH43, and pKH45, plasmid pKH7 was digested with the restriction enzymesSalI-XhoI (pKH23),CelII-SalI (pKH33),BstEII-NruI (pKH34),NruI-CelII (pKH35),BspEI-NheI (pKH43), orBspEI-BstEII (pKH45), respectively. The resulting 5′-overhangs were filled in using the Klenow fragment of E. coli DNA polymerase I and dNTPs (except in the case of pKH23), and the plasmids were religated. Plasmids pKH44 and pKH46 were generated similarly to pKH43 and pKH45, respectively, except that they were derived from pKH14. Plasmid pJDSac was constructed by introducing the 2.4-kb SacI-SacI fragment from pRSMot1Myc into YEp352GAL (47Benton B.M. Eng W.K. Dunn J.J. Studier F.W. Sternglanz R. Fisher P.A. Mol. Cell. Biol. 1990; 10: 353-360Crossref PubMed Scopus (57) Google Scholar). To create pKH1, pRSMot1Myc was digested withNheI and HpaI, treated with mung bean nuclease (Stratagene), and religated. Plasmid pKH24 was made by swapping the 2.4-kb XhoI-BspEI fragment from pKH1 into pKH7. Plasmids pKH36, pKH37, pKH41, pKH42, and pKH47 were generated by swapping the 3-kb D1408N mutation-containing NdeI fragment from pKH14 into pKH24, pKH34, pKH33, pKH35, and pJDSac, respectively. pKH25 was constructed by using PCR to introduce a XhoI site just 3′ to codon 1261 and then deleting theXhoI-XhoI fragment corresponding to the sequences coding for amino acids 1262–1867, but retaining the c-Myc epitope tag. A 472-base pair fragment of MOT1 was amplified using primers KHP5 and KHP6, digested with XhoI and AflII, and ligated to XhoI-AflII-digested pKH7, to generate pKH25. All deletions of MOT1 were sequenced to confirm that the junctions were in-frame and at the positions expected. MOT1and MOT1(D1408N) were transcribed from the T3 promoter using plasmids pKH21 and pKH22, respectively. pKH21 was constructed by swapping the 2.4-kbSacI fragment from pKH7 into pKH2 (28Auble D.T. Hansen K.E. Mueller C.G. Lane W.S. Thorner J. Hahn S. Genes Dev. 1994; 8: 1920-1934Crossref PubMed Scopus (277) Google Scholar). pKH22 was made in an identical manner, except that the SacI fragment was derived from pKH14. As a negative control, a construct expressingRAD3 from the T3 promoter (48Bardwell L. Bardwell A.J. Feaver W.J. Svejstrup J.Q. Kornberg R.D. Friedberg E.C. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 3926-3930Crossref PubMed Scopus (37) Google Scholar) was transcribed.SPT15 (yeast TBP), with an in-frame N-terminal (His)6 tag, was transcribed from the T7 promoter in plasmid pKH19, which was constructed (39Hansen K. Biochemical and Genetic Characterization of Mot1 and Its Interaction with Components of the Basal Transcription Complex in Saccharomyces cerevisiae Ph.D. thesis. University of California, Berkeley1996Google Scholar) by inserting a 1.5-kbNdeI-BamHI fragment containing theSPT15 coding sequence from pM1 (gift of Robert Tjian, University of California, Berkeley) intoNdeI-BamHI-digested pET15b (Novagen). Three TBP mutants, spt15(K133L K138L),spt15(K133L K145L), andspt15(K138T Y139A), were expressed from plasmids pKH39, pKH40, and pKH48, respectively, which were constructed by transferring the Bsu36I-BamHI fragment containing the appropriate portion of the SPT15 open reading frame (ORF) from pT7-IID(K133L,K138L), pT7-IID(K133L,K145L) (41Buratowski S. Zhou H. Science. 1992; 255: 1130-1132Crossref PubMed Scopus (82) Google Scholar), and TBP(N2-1) (49Stargell L.A. Struhl K. Science. 1995; 269: 75-78Crossref PubMed Scopus (99) Google Scholar), respectively, into pKH19. pKH38 is identical to pKH39 and pKH40, except that it expresses wild-type SPT15 and was generated by swapping the Bsu36I-BamHI fragment from pT7-IID (41Buratowski S. Zhou H. Science. 1992; 255: 1130-1132Crossref PubMed Scopus (82) Google Scholar) into pKH19. The NdeI-BamHI fragment from pT7-IID(R105H) (gift of Dr. Steve Buratowski, Harvard Medical School) was transferred into NdeI-BamHI- digested pKH19 to yield plasmid pKH49, which expressesspt15(R105H) from the T3 promoter. pKH38 (instead of pKH19) was used to prepare wild-type TBP for those experiments in which normal TBP was compared with TBP mutants because pKH38 was constructed in parallel with the plasmids expressing the TBP mutants. To express MOT1, mot1(Δ25-243), mot1(Δ494-801), mot1(Δ802-1259), mot1(Δ1262-1867), andmot1(Δ1089-1867) by in vitro transcription, PCR was used to amplify sequences containing the MOT1 deletion mutants from plasmids pKH7, pKH43, pKH34, pKH35, pKH25, and pKH23, respectively. In each case, a 5′-primer (KHP22) that corresponds to a segment of the T3 promoter fused to theMOT1 translation start site was used in combination with a primer (T7) that anneals 3′ to the MOT1 ORF. C-terminal deletions (see Fig. 4) were constructed by linearizing plasmid pKH2 with restriction enzymes at sites within the ORF, allowing for run-off transcription. In the case of enzymes leaving 3′-overhangs, the 3′-exonuclease activity of T4 DNA polymerase was used to digest the overhanging ends to flush ends, so as to prevent spurious initiation by T3 RNA polymerase. The deletions and restriction enzymes used are as follows: Δ1823–1867, BstXI; Δ1676–1867,EcoNI; Δ1386–1867, NdeI; Δ1280–1867,HpaI; Δ1258–1867, CelII; Δ1203–1867,BspBI; Δ1167–1867, PvuI; Δ1120–1867,AflII; Δ1087–1867, SalI; Δ842–1867,MunI; and Δ800–1867, NruI. Most N-terminal deletions were made by PCR using T3 promoter-containing primers. The resulting PCR products were then transcribed directly. Themot1(Δ1-897) fragment was made with primers KHP12 and KHP14, using linearized pKH2 as the template. Themot1(Δ1-1253),mot1(Δ1-655), andmot1(Δ1-513) fragments were made in a similar manner except that primers, KHP13, KHP15, and KHP16, respectively, were used in place of KHP12. Primers KHP13 and KHP14 were used to make mot1(Δ1-1253). Themot1(Δ1-100) fragment was made using primers T7 and KHP23. Themot1(Δ1-1100) fragment was transcribed from plasmid pKH27 (39Hansen K. Biochemical and Genetic Characterization of Mot1 and Its Interaction with Components of the Basal Transcription Complex in Saccharomyces cerevisiae Ph.D. thesis. University of California, Berkeley1996Google Scholar), which was made by digesting pKH2 with XhoI and SalI and religating the vector. The PCR product generated using primers KHP9 and JDM6 with pKH2 as the template was digested with XhoI and SalI and introduced into pKH2 digested with the same enzymes to create plasmid pKH29, which expresses mot1(Δ1-1026) from the T3 RNA polymerase promoter. Proteins to be used in coimmunoprecipitation experiments were produced by coupled in vitro transcription and translation in the presence of [35S]Met (PerkinElmer Life Sciences) using the PromegaTNTTM Coupled Reticulocyte Lysate System, according to the manufacturer's directions. To increase yield, 0.2 mg/ml yeast tRNA (Sigma) was added to each translation mixture. Prior to use, PCR-derived or linearized DNA templates were extracted with phenol:chloroform:isoamyl alcohol (25:24:1) and precipitated with ethanol. After translation, proteins were partially purified by precipitation with 50% ammonium sulfate (50Bardwell L. Cooper A.J. Friedberg E.C. Mol. Cell. Biol. 1992; 12: 3041-3049Crossref PubMed Scopus (73) Google Scholar), which also allowed for quantification of translation yield as described in detail elsewhere (51Bardwell L. Cook J.G. Chang E.C. Cairns B.R. Thorner J. Mol. Cell. Biol. 1996; 16: 3637-3650Crossref PubMed Scopus (134) Google Scholar). Briefly, the concentration of translated protein can be estimated based on the percent incorporation of [35S]Met, the number of Met residues known to be present in the sequence of the protein, the molecular weight of the protein, and the concentration of unlabeled methionine in the extract (∼5–10 μm). The ammonium sulfate precipitates were resuspended in Buffer A (20 mm Tris-HCl (pH 7.5), 75 mm potassium acetate, 10 mm MgCl2, 1 mm EDTA, 1 mm dithiothreitol (DTT), 0.1% Tween 20, and 12.5% glycerol) at a volume equal to the starting volume. Immunoprecipitations were performed following the procedures described (51Bardwell L. Cook J.G. Chang E.C. Cairns B.R. Thorner J. Mol. Cell. Biol. 1996; 16: 3637-3650Crossref PubMed Scopus (134) Google Scholar). Radiolabeled proteins (∼0.5 pmol each), prepared by coupled in vitro transcription and translation as described above, were mixed in a final volume of 200 μl of Buffer A, incubated for 30 min at 4 °C, and then clarified by centrifugation at maximum speed in a microcentrifuge at 4 °C for 15 min. In some experiments (see Fig. 1), unlabeled yeast TBP (yTBP), which was expressed in and purified from E. coli according to the method of Ref. 52Kim Y. Geiger J.H. Hahn S. Sigler P.B. Nature. 1993; 365: 512-520Crossref PubMed Scopus (1016) Google Scholar, was used. After clarification, the resulting supernatant solution was withdrawn and mixed with 15 μl of a 50:50 slurry of protein A-agarose beads (Oncogene Sciences) in Buffer A and 2 μl of polyclonal anti-yTBP antibodies (gift of Dr. Grace Gill, Harvard Medical School) in a 1.5-ml microcentrifuge tube. After incubation at 4 °C for 1 h on a roller drum, the agarose beads were collected by centrifugation for 5 s in a microcentrifuge. The supernatant solution was removed by aspiration using a 25-gauge needle, and the pellets were washed 3 times with 1 ml of ice-cold Buffer A. The bead-bound immune complexes were resuspended in SDS sample buffer, boiled, and resolved on a 10% SDS-polyacrylamide gel, along with a lane containing 14C-labeled molecular weight markers (Amersham Pharmacia Biotech). After electrophoresis, gels were fixed for 30 min in 10% methanol and 10% acetic acid, dried under vacuum, and analyzed by autoradiography using x-ray film (Kodak Biomax MR) or quantified using a PhosphorImagerTM (Molecular Dynamics). Cultures of strain W303-1A harboring plasmids containing the various MOT1Myc derivatives were grown to an approximate density of A 600 nm = 0.7 in SCRaf medium lacking uracil, induced by addition of Gal, and grown for an additional 3 h. Cells were harvested by centrifugation and washed once with ice-cold lysis buffer (50 mm Tris-HCl (pH 7.5), 100 mm NaCl, 5 mm EDTA, 5 mm EGTA, 2 mmphenylmethylsulfonyl fluoride, 20 μg/ml pepstatin A, 1 mmDTT). All subsequent steps were carried out at 4 °C. An amount of cells (equivalent to 10 A 600 nm units) was resuspended in 200 μl of lysis buffer and lysed by vortex mixing with glass beads as described in detail (53Ma D. Cook J.G. Thorner J. Mol. Biol. Cell. 1995; 6: 889-909Crossref PubMed Scopus (68) Google Scholar). Unbroken cells and large cell debris were removed by low speed centrifugation (2000 rpm for 5 min in a Sorvall SS-34). For immunodetection of Mot1, a sample of the lysate equivalent to 30 μg
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