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• W2086065602 abstract "Essentially all nuclear eukaryotic gene transcription depends upon the function of the transcription factor TATA-binding protein (TBP). Here we show that the abundant, multifunctional DNA binding transcription factor repressor activator protein Rap1p interacts directly with TBP. TBP-Rap1p binding occurs efficiently in vivo at physiological expression levels, and in vitro analyses confirm that this is a direct interaction. The DNA binding domains of the two proteins mediate interaction between TBP and Rap1p. TBP-Rap1p complex formation inhibits TBP binding to TATA promoter DNA. Alterations in either Rap1p or TBP levels modulate mRNA gene transcription in vivo. We propose that Rap1p represents a heretofore unrecognized regulator of TBP. Essentially all nuclear eukaryotic gene transcription depends upon the function of the transcription factor TATA-binding protein (TBP). Here we show that the abundant, multifunctional DNA binding transcription factor repressor activator protein Rap1p interacts directly with TBP. TBP-Rap1p binding occurs efficiently in vivo at physiological expression levels, and in vitro analyses confirm that this is a direct interaction. The DNA binding domains of the two proteins mediate interaction between TBP and Rap1p. TBP-Rap1p complex formation inhibits TBP binding to TATA promoter DNA. Alterations in either Rap1p or TBP levels modulate mRNA gene transcription in vivo. We propose that Rap1p represents a heretofore unrecognized regulator of TBP. Eukaryotic gene transcription is controlled through the concerted action of DNA-binding transfactors, proteins that functionally interact with the transcription machinery to turn genes on and off. For mRNA-encoding genes the transcription machinery is composed of the general transcription factors (GTFs) 2The abbreviations used are:GTFgeneral transcription factorTBPTATA-binding proteinRap1prepressor activator proteinPICpre-initiation complexTAFTATA-binding protein-associated factorRPribosomal proteinDBDDNA binding domainUASupstream activating sequenceGSTglutathione S-transferaseaaamino acidTANDTAF1 N-terminal domainWTwild typeTAPtandem affinity purificationAd2 MLPadenovirus type 2 major late promoterWCEwhole cell extractIPimmunoprecipitationco-IPco-immunoprecipitationChIPchromatin immunoprecipitationTETtetracyclineAITactivator-independent transcriptionTFtranscription factorRNAPRNA polymeraseHAhemagglutinin. 2The abbreviations used are:GTFgeneral transcription factorTBPTATA-binding proteinRap1prepressor activator proteinPICpre-initiation complexTAFTATA-binding protein-associated factorRPribosomal proteinDBDDNA binding domainUASupstream activating sequenceGSTglutathione S-transferaseaaamino acidTANDTAF1 N-terminal domainWTwild typeTAPtandem affinity purificationAd2 MLPadenovirus type 2 major late promoterWCEwhole cell extractIPimmunoprecipitationco-IPco-immunoprecipitationChIPchromatin immunoprecipitationTETtetracyclineAITactivator-independent transcriptionTFtranscription factorRNAPRNA polymeraseHAhemagglutinin. TFIIA, TFIIB, TFIID, TFIIE, TFIIF, TFIIH, and RNA polymerase (RNAP) II. DNA-bound transactivators stimulate the GTFs and RNAP II to form pre-initiation complexes (PIC) on cis-linked promoters (1Szutorisz H. Dillon N. Tora L. Trends Biochem. Sci. 2005; 30: 593-599Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). TBP recruitment to the TATA box of mRNA gene promoters is a critical, and likely rate-limiting step in nucleating this process (2Klein C. Struhl K. Science. 1994; 266: 280-282Crossref PubMed Scopus (106) Google Scholar, 3Li X.Y. Bhaumik S.R. Green M.R. Science. 2000; 288: 1242-1244Crossref PubMed Scopus (156) Google Scholar, 4Kuras L. Struhl K. Nature. 1999; 399: 609-613Crossref PubMed Scopus (399) Google Scholar). TBP is chaperoned to promoters by a number of proteins, and in the case of mRNA-encoding genes, TFIID is the predominant protein complex that serves this function. TFIID has 15 evolutionarily conserved subunits, TBP and 14 TBP-associated factors (TAFs) (5Sanders S.L. Garbett K.A. Weil P.A. Mol. Cell. Biol. 2002; 22: 6000-6013Crossref PubMed Scopus (89) Google Scholar, 6Tora L. Genes Dev. 2002; 16: 673-675Crossref PubMed Scopus (193) Google Scholar). The TATA box promoter element of mRNA-encoding genes is bound via the TBP subunit of TFIID through a process facilitated by TAFs (7Burke T.W. Kadonaga J.T. Genes Dev. 1996; 10: 711-724Crossref PubMed Scopus (324) Google Scholar, 8Metcalf C.E. Wassarman D.A. J. Biol. Chem. 2006; 281: 30015-30023Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar, 9Lee D.H. Gershenzon N. Gupta M. Ioshikhes I.P. Reinberg D. Lewis B.A. Mol. Cell. Biol. 2005; 25: 9674-9686Crossref PubMed Scopus (84) Google Scholar, 10Chalkley G.E. Verrijzer C.P. EMBO J. 1999; 18: 4835-4845Crossref PubMed Scopus (176) Google Scholar). general transcription factor TATA-binding protein repressor activator protein pre-initiation complex TATA-binding protein-associated factor ribosomal protein DNA binding domain upstream activating sequence glutathione S-transferase amino acid TAF1 N-terminal domain wild type tandem affinity purification adenovirus type 2 major late promoter whole cell extract immunoprecipitation co-immunoprecipitation chromatin immunoprecipitation tetracycline activator-independent transcription transcription factor RNA polymerase hemagglutinin. general transcription factor TATA-binding protein repressor activator protein pre-initiation complex TATA-binding protein-associated factor ribosomal protein DNA binding domain upstream activating sequence glutathione S-transferase amino acid TAF1 N-terminal domain wild type tandem affinity purification adenovirus type 2 major late promoter whole cell extract immunoprecipitation co-immunoprecipitation chromatin immunoprecipitation tetracycline activator-independent transcription transcription factor RNA polymerase hemagglutinin. TBP is required for nearly all nuclear gene transcription as it is an integral subunit of the RNAP I-, II-, and III-specific initiation factors SL1, TFIID, and TFIIIB (11Hernandez N. Genes Dev. 1993; 7: 1291-1308Crossref PubMed Scopus (561) Google Scholar). Consequently, it is not surprising that TBP function is subject to tight regulation by non-GTF transcription factors such as NC2, Mot1p, and SAGA and NOT that all directly bind TBP and modulate its activity (12Pugh B.F. Gene (Amst.). 2000; 255: 1-14Crossref PubMed Scopus (154) Google Scholar, 13Pereira L.A. Klejman M.P. Timmers H.T. Gene (Amst.). 2003; 315: 1-13Crossref PubMed Scopus (43) Google Scholar). Yeast Rap1p is encoded by a single-copy essential gene and plays multiple roles in vivo (14Shore D. Trends Genet. 1994; 10: 408-412Abstract Full Text PDF PubMed Scopus (261) Google Scholar, 15Morse R.H. Trends Genet. 2000; 16: 51-53Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar, 16Rudra D. Warner J.R. Genes Dev. 2004; 18: 2431-2436Crossref PubMed Scopus (178) Google Scholar, 17Pina B. Fernandez-Larrea J. Garcia-Reyero N. Idrissi F.Z. Mol. Genet. Genomics. 2003; 268: 791-798Crossref PubMed Scopus (63) Google Scholar). Rap1p is a key transactivator of over 300 co-regulated genes, including the ribosomal protein (RP) (18Lieb J.D. Liu X. Botstein D. Brown P.O. Nat. Genet. 2001; 28: 327-334Crossref PubMed Scopus (548) Google Scholar) and glycolytic enzyme-encoding genes (19Buchman A.R. Lue N.F. Kornberg R.D. Mol. Cell. Biol. 1988; 8: 5086-5099Crossref PubMed Scopus (202) Google Scholar, 20Drazinic C.M. Smerage J.B. Lopez M.C. Baker H.V. Mol. Cell. Biol. 1996; 16: 3187-3196Crossref PubMed Scopus (46) Google Scholar), and it drives 40% of all mRNA gene transcription initiation events in actively growing cells (18Lieb J.D. Liu X. Botstein D. Brown P.O. Nat. Genet. 2001; 28: 327-334Crossref PubMed Scopus (548) Google Scholar). Additionally, Rap1p participates in transcriptional repression (21Kyrion G. Liu K. Liu C. Lustig A.J. Genes Dev. 1993; 7: 1146-1159Crossref PubMed Scopus (239) Google Scholar), telomere length modulation (22Marcand S. Gilson E. Shore D. Science. 1997; 275: 986-990Crossref PubMed Scopus (424) Google Scholar), recombination (23Stavenhagen J.B. Zakian V.A. Genes Dev. 1998; 12: 3044-3058Crossref PubMed Scopus (29) Google Scholar), and chromatin barrier function (24Bi X. Broach J.R. Genes Dev. 1999; 13: 1089-1101Crossref PubMed Scopus (117) Google Scholar, 25Fourel G. Miyake T. Defossez P.A. Li R. Gilson E. J. Biol. Chem. 2002; 277: 41736-41743Abstract Full Text Full Text PDF PubMed Scopus (45) Google Scholar). The only essential domain of Rap1p is its DNA-binding domain (DBD), which is composed of two Myb-like motifs. Yeast expressing only the Rap1p DBD grow, albeit extremely slowly (26Graham I.R. Haw R.A. Spink K.G. Halden K.A. Chambers A. Mol. Cell. Biol. 1999; 19: 7481-7490Crossref PubMed Scopus (41) Google Scholar). Deletion of the N-terminal BRCT-DNA bending domain has minimal effects on viability (27Muller T. Gilson E. Schmidt R. Giraldo R. Sogo J. Gross H. Gasser S.M. J. Struct. Biol. 1994; 113: 1-12Crossref PubMed Scopus (44) Google Scholar, 28Del Vescovo V. De Sanctis V. Bianchi A. Shore D. Di Mauro E. Negri R. J. Mol. Biol. 2004; 338: 877-893Crossref PubMed Scopus (12) Google Scholar), whereas removal of sequences C-terminal to the DBD results in slow growth (26Graham I.R. Haw R.A. Spink K.G. Halden K.A. Chambers A. Mol. Cell. Biol. 1999; 19: 7481-7490Crossref PubMed Scopus (41) Google Scholar). A C-terminal activation domain, fused to a heterologous DBD, weakly activates transcription of a reporter gene (29Hardy C.F. Balderes D. Shore D. Mol. Cell. Biol. 1992; 12: 1209-1217Crossref PubMed Scopus (75) Google Scholar); by contrast, lexA full-length Rap1p fusions fail to activate (30Zhao Y. McIntosh K.B. Rudra D. Schawalder S. Shore D. Warner J.R. Mol. Cell. Biol. 2006; 26: 4853-4862Crossref PubMed Scopus (81) Google Scholar). The C-terminal silencing domain represses mating-type, telomere-proximal, and RP genes under certain circumstances (31Moretti P. Freeman K. Coodly L. Shore D. Genes Dev. 1994; 8: 2257-2269Crossref PubMed Scopus (461) Google Scholar, 32Moretti P. Shore D. Mol. Cell. Biol. 2001; 21: 8082-8094Crossref PubMed Scopus (82) Google Scholar). Finally, a 34-amino acid (aa) Tox domain inhibits cell growth when overexpressed fused to the DBD (17Pina B. Fernandez-Larrea J. Garcia-Reyero N. Idrissi F.Z. Mol. Genet. Genomics. 2003; 268: 791-798Crossref PubMed Scopus (63) Google Scholar, 33Freeman K. Gwadz M. Shore D. Genetics. 1995; 141: 1253-1262Crossref PubMed Google Scholar, 34Idrissi F.Z. Garcia-Reyero N. Fernandez-Larrea J.B. Pina B. J. Biol. Chem. 2001; 276: 26090-26098Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar, 35De Sanctis V. La Terra S. Bianchi A. Shore D. Burderi L. Di Mauro E. Negri R. J. Mol. Biol. 2002; 318: 333-349Crossref PubMed Scopus (12) Google Scholar). Several mechanisms have been proposed for how Rap1p controls gene transcription. First, Rap1p-upstream activating sequence/enhancer (UASRAP1) binding may exclude nucleosomes and stimulate transcription by increasing local DNA ciselement accessibility (30Zhao Y. McIntosh K.B. Rudra D. Schawalder S. Shore D. Warner J.R. Mol. Cell. Biol. 2006; 26: 4853-4862Crossref PubMed Scopus (81) Google Scholar, 36Yu L. Morse R.H. Mol. Cell. Biol. 1999; 19: 5279-5288Crossref PubMed Scopus (89) Google Scholar). Rap1p binds nucleosomal RAP1 sites efficiently in vitro (37Rossetti L. Cacchione S. De Menna A. Chapman L. Rhodes D. Savino M. J. Mol. Biol. 2001; 306: 903-913Crossref PubMed Scopus (29) Google Scholar), and in vivo UASRAP1 DNA is deficient in nucleosomes (30Zhao Y. McIntosh K.B. Rudra D. Schawalder S. Shore D. Warner J.R. Mol. Cell. Biol. 2006; 26: 4853-4862Crossref PubMed Scopus (81) Google Scholar, 38Sekinger E.A. Moqtaderi Z. Struhl K. Mol. Cell. 2005; 18: 735-748Abstract Full Text Full Text PDF PubMed Scopus (270) Google Scholar, 39Segal E. Fondufe-Mittendorf Y. Chen L. Thastrom A. Field Y. Moore I.K. Wang J.P. Widom J. Nature. 2006; 442: 772-778Crossref PubMed Scopus (1180) Google Scholar, 40Bernstein B.E. Liu C.L. Humphrey E.L. Perlstein E.O. Schreiber S.L. Genome Biol. 2004; 5: R62Crossref PubMed Google Scholar, 41Ioshikhes I.P. Albert I. Zanton S.J. Pugh B.F. Nat. Genet. 2006; 38: 1210-1215Crossref PubMed Scopus (260) Google Scholar, 42Lee C.K. Shibata Y. Rao B. Strahl B.D. Lieb J.D. Nat. Genet. 2004; 36: 900-905Crossref PubMed Scopus (564) Google Scholar) although some nucleosomal RAP1 sites are only bound conditionally (43Zanton S.J. Pugh B.F. Genes Dev. 2006; 20: 2250-2265Crossref PubMed Scopus (86) Google Scholar, 44Buck M.J. Lieb J.D. Nat. Genet. 2006; 38: 1446-1451Crossref PubMed Scopus (73) Google Scholar). Second, Rap1p functionally interacts with other DNA binding factors (i.e. Gcr1p-Gcr2p, Abf1p, Reb1p, Fhl1p-Ifh1p, Sfp1p, and Hmo1p) (45Deminoff S.J. Santangelo G.M. Genetics. 2001; 158: 133-143PubMed Google Scholar, 46Wade J.T. Hall D.B. Struhl K. Nature. 2004; 432: 1054-1058Crossref PubMed Scopus (162) Google Scholar, 47Rudra D. Zhao Y. Warner J.R. EMBO J. 2005; 24: 533-542Crossref PubMed Scopus (138) Google Scholar, 48Martin D.E. Soulard A. Hall M.N. Cell. 2004; 119: 969-979Abstract Full Text Full Text PDF PubMed Scopus (351) Google Scholar, 49Marion R.M. Regev A. Segal E. Barash Y. Koller D. Friedman N. O'Shea E.K. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 14315-14322Crossref PubMed Scopus (268) Google Scholar, 50Jorgensen P. Rupes I. Sharom J.R. Schneper L. Broach J.R. Tyers M. Genes Dev. 2004; 18: 2491-2505Crossref PubMed Scopus (491) Google Scholar, 51Hall D.B. Wade J.T. Struhl K. Mol. Cell. Biol. 2006; 26: 3672-3679Crossref PubMed Scopus (107) Google Scholar, 52Kasahara K. Ohtsuki K. Ki S. Aoyama K. Takahashi H. Kobayashi T. Shirahige K. Kokubo T. Mol. Cell. Biol. 2007; 27: 6686-6705Crossref PubMed Scopus (60) Google Scholar), and with multisubunit transcriptional coregulators such as NuA4/Esa1p, SWI/SNF, and TFIID (34Idrissi F.Z. Garcia-Reyero N. Fernandez-Larrea J.B. Pina B. J. Biol. Chem. 2001; 276: 26090-26098Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar, 53Mencia M. Moqtaderi Z. Geisberg J.V. Kuras L. Struhl K. Mol. Cell. 2002; 9: 823-833Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar, 54Reid J.L. Iyer V.R. Brown P.O. Struhl K. Mol. Cell. 2000; 6: 1297-1307Abstract Full Text Full Text PDF PubMed Scopus (248) Google Scholar). We have recently shown that Rap1p directly binds several TAF subunits of TFIID (55Garbett K.A. Tripathi M.K. Cencki B. Layer J.H. Weil P.A. Mol. Cell. Biol. 2007; 27: 297-311Crossref PubMed Scopus (62) Google Scholar). Third, Rap1p interacts with Rif and Sir proteins, which contribute to transcriptional repression of telomere-proximal and HML/HMR genes, as well as telomere-length modulation (31Moretti P. Freeman K. Coodly L. Shore D. Genes Dev. 1994; 8: 2257-2269Crossref PubMed Scopus (461) Google Scholar, 32Moretti P. Shore D. Mol. Cell. Biol. 2001; 21: 8082-8094Crossref PubMed Scopus (82) Google Scholar, 56Luo K. Vega-Palas M.A. Grunstein M. Genes Dev. 2002; 16: 1528-1539Crossref PubMed Scopus (185) Google Scholar, 57Wotton D. Shore D. Genes Dev. 1997; 11: 748-760Crossref PubMed Scopus (356) Google Scholar). Despite the fact that Rap1p has been studied extensively, the exact molecular mechanisms through which these myriad Rap1p protein-protein interactions lead to transcription activation and repression remain to be deciphered. In this report we describe the results of our continuing investigations into how Rap1p interacts with the transcription machinery. We have found that Rap1p directly and specifically binds TBP, and have molecularly dissected this interaction. We discuss the implications of Rap1p-TBP binding vis à vis the regulation of TBP activity. Plasmids, Protein Purification, Yeast Strains, Yeast Cell Growth, and Manipulation—For TBP, His6-TBP (58Banik 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, 59Bai Y. Perez G.M. Beechem J.M. Weil P.A. Mol. Cell. Biol. 1997; 17: 3081-3093Crossref PubMed Scopus (61) Google Scholar) was used; other TBP variants were cloned in pBG101 as BamHI-EcoRI inserts for the production of His6-glutathione S-transferase (GST)-TBP and TBP. Rap1p variants were cloned in pET42a as EcoRI-XhoI inserts for the production of GST-Rap1p; His6-GST-Rap1p and Rap1p variants were produced from Rap1p forms cloned in pBGMB1 (a version of pBG101 with BamHI and EcoRI sites inverted) as EcoRI-XhoI inserts. For TFIIA, pToa1 and pToa2 (60Tan S. Hunziker Y. Sargent D.F. Richmond T.J. Nature. 1996; 381: 127-151Crossref PubMed Scopus (257) Google Scholar) were used; for TFIIB, His6-tagged (61Wu W.H. Hampsey M. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 2764-2769Crossref PubMed Scopus (40) Google Scholar); for NC2, His6-tagged Ncb1p and Ncb2p (62Kim S. Cabane K. Hampsey M. Reinberg D. Mol. Cell. Biol. 2000; 20: 2455-2465Crossref PubMed Scopus (19) Google Scholar); and for Taf1p TAND (TAF1 N-terminal domain), the TAF1 open reading frame sequence encoding amino acids (aa) 1-380 was PCR-amplified with NheI (5′) and EcoRI (3′) ends and cloned into pET28a. TFIIA, TFIIB, His6-TBP, His6-TAND, and NC2 proteins were produced as described as listed above. His6-GST-TBP and His6-GST-Rap1p variants were expressed and prepared as for His6-TBP. His6-GST fusion variants were bound to glutathione-agarose beads, glutathione-eluted, and treated with His6-tagged precission protease on Ni-nitriloacetic acid-agarose beads for 1 h at room temperature and centrifuged to remove the beads. Supernatants were collected, analyzed for protein, and stored at -80 °C. Tandem affinity purification (TAP)-tagged wild type (WT), W303a (MATα ade2-1 leu2-3,112 ura3-1 his3-11,15 trp1-1 can1-100 ssd1-1); TAP-Rap1p, W303a (TAP-RAP1::TRP1); Rap1p-TAP, W303a (RAP1-TAP::HIS3). MYC-tagged WT, W303a (MATa ade2-1 leu2-3,112 ura3-1 his3-11,15 trp1-1 can1-100 ssd1-1); MYC-Rap1p, W303a (RAP1-MYC18::TRP1) were used. Rap1p knockdown, control strain, ZMY60 (MATa ura3-52 trp1-Δ1 ade2-101 pACE1-UBR1 pACE1-ROX1) (63Moqtaderi Z. Bai Y. Poon D. Weil P.A. Struhl K. Nature. 1996; 383: 188-191Crossref PubMed Scopus (250) Google Scholar); Rap1p knockdown strain LEV391 ZMY60(rap1-(Δ)::KANr (KANr-ANB-UB-R-lacI-4HA-RAP1) (64Pardo B. Marcand S. EMBO J. 2005; 24: 3117-3127Crossref PubMed Scopus (123) Google Scholar) were also used. RAP1 variants were cloned as EcoRI-ClaI inserts either in pESC vectors (UASGAL-dependent expression), in pRS415 vector (ADH1-enhancer/promoter) for the production of Rap1p-FLAG forms, or pRS414, controlled by RAP1 enhancer/promoter and RAP1 terminator. Wild type TBP was cloned in the same pESC vector ± RAP1 as an XhoI-flanked insert for the expression of MYC-TBP (Fig. 5, B and C); TBP variants (Fig. 6) were cloned as BamHI-XhoI inserts in pRS413 (ADH1-dependent expression) for the production of HA3-TBP. Yeast were grown, transformed, plated, and subjected to 5-fluoroorotic acid plasmid shuffle growth tests using standard methods.FIGURE 6Rap1p DBD modulates mRNA reporter gene transcription in vivo. A, yeast strain W303a, containing one of two plasmid-born lacZ reporter genes (pADH1-lacZ, pCYC1-lacZ), was transformed with empty vector (-) or vector expressing the indicated HA-tagged forms of TBP. Top, -fold activation of β-galactosidase induction in cells overexpressing either no TBP, or the indicated forms of TBP; expression was from pADH1-lacZ (light gray) or pCYC1-lacZ (defined as 1.0; dark gray bars) reporters. Data from three simultaneous independent experiments is shown along with S.E. Bottom, expression of TBP variants as measured by immunoblotting WCEs with anti-HA monoclonal antibody. B, yeast strain W303a, carrying a URA3-marked pADH1-lacZ reporter was transformed with a HIS3-marked plasmid, which was either empty (-) or expressing HA-tagged WT, R98E, T124R, V161R, R196E, N69S, or L205K mutants of TBP, and/or a LEU2-marked plasmid, either empty (+Vector; white bars), or expressing the FLAG-tagged ΔTox (dark gray bars), DBD (black bars), or ΔDBD (light gray bars) Rap1p variants. β-Galactosidase activity was measured and plotted as -fold activation by TBP overexpression relative to the amount of β-galactosidase expressed in cells carrying the empty vectors. The effect of Rap1p variants upon TBP-induced AIT was calculated as above; right, immunoblot showing protein expression levels of the Rap1p and TBP variants.View Large Image Figure ViewerDownload Hi-res image Download (PPT) DNase I Footprinting—DNase I footprinting using Ad2 MLP (major late promoter) (-250 to +195 relative to the +1 transcription start site) and RPS1A (-420 to +23 relative to the A+1TG Rps1p methionine codon) probes was performed as described (5Sanders S.L. Garbett K.A. Weil P.A. Mol. Cell. Biol. 2002; 22: 6000-6013Crossref PubMed Scopus (89) Google Scholar). GST Pulldown Assay—100-μl binding reactions were performed in binding buffer: 20 mm HEPES-KOH (pH 7.6), 10% glycerol, 70 mm potassium acetate, 0.1 mm EDTA, 1 mm dithiothreitol, 5 mm MgAc, 0.01% Nonidet P-40, plus 10 μl of beads (300 ng of GST fusion protein/reaction), and the proteins to be tested for binding. Reactions were mixed 30 min at room temperature, then beads were washed 3 times with binding buffer, resuspended in 20 μl of SDS-PAGE sample buffer, heat denatured, and analyzed by SDS-PAGE on NuPAGE 4-12% polyacrylamide gels followed by Sypro Ruby staining or immunoblotting. TBP Cross-linking—TBP (100 ng/μl) in 19 μl of 20 mm HEPES-KOH (pH 7.6), 10% glycerol, 150 mm NaAc, 5 mm MgAc, and 0.05% Nonidet P-40 was incubated for 30 min at room temperature, then 1 μl of 20% formaldehyde, 20 mm HEPES-KOH (pH 7.6), 150 mm sodium acetate was added and incubation continued for 15 min at room temperature. Cross-linking was quenched by addition of 3 μl of 2.5 m glycine and incubation for 5 min at room temperature. 20 μl of SDS-PAGE sample buffer was added and samples were loaded, without heating, on 4-12% NuPAGE gels followed by Sypro Ruby staining or immunoblotting. Whole Cell Extract (WCE) Preparation, Immunoprecipitations (IPs), and Immunoblotting—WCE preparation, IPs, immunoblotting, and chemiluminescent detection were performed as described (65Sanders S.L. Jennings J. Canutescu A. Link A.J. Weil P.A. Mol. Cell. Biol. 2002; 22: 4723-4738Crossref PubMed Scopus (260) Google Scholar). β-Galactosidase Assay—Freshly transformed cells were used. Colonies were used to inoculate 5 ml of selective media cultures, grown to 1 A600/ml, and cells tested for β-galactosidase activity as described by Blair and Cullen (72Blair W.S. Cullen B.R. Mol. Cell. Biol. 1997; 17: 2888-2896Crossref PubMed Scopus (17) Google Scholar). Primer Extension and Chromatin Immunoprecipitation (ChIP) Assays—Primer extension (67Schroeder S.C. Wang C.K. Weil P.A. J. Biol. Chem. 1994; 269: 28335-28346Abstract Full Text PDF PubMed Google Scholar) and ChIP assays were performed essentially as described (55Garbett K.A. Tripathi M.K. Cencki B. Layer J.H. Weil P.A. Mol. Cell. Biol. 2007; 27: 297-311Crossref PubMed Scopus (62) Google Scholar) except ChIP PCR products were fractionated by non-denaturing polyacrylamide gel electrophoresis in 1× TBE buffer and DNA quantified by staining with Sypro Gold (Invitrogen). While conducting footprinting experiments to examine the binding of yeast TFIID to a battery of Rap1p-dependent and Rap1p-independent genes, control reactions showed that Rap1p addition blocked TBP-TATA DNA binding. Given the central role of TBP in nuclear gene transcription, the potency of inhibition of TBP-DNA binding by nanomolar concentrations of Rap1p, the nuclear abundance, and high concentration of both proteins (≥100 μm), we examined this phenomenon in more detail. Rap1p Inhibits TATA DNA Binding by TBP—The inhibitory effect of Rap1p upon the binding of TBP (see Fig. 1A for purity of proteins) to the Adenovirus 2 major late promoter (Ad2 MLP), a heterologous DNA devoid of RAP1 sites, is shown in Fig. 1B (left panel, Ad2 MLP). As expected, TBP bound to the Ad2 MLP TATA box (lane 1 versus 4), and at the TBP concentration used here, also bound to two TA-rich regions up- and downstream of TATA (TA-I, TA-II) (5Sanders S.L. Garbett K.A. Weil P.A. Mol. Cell. Biol. 2002; 22: 6000-6013Crossref PubMed Scopus (89) Google Scholar, 66Coleman R.A. Pugh B.F. J. Biol. Chem. 1995; 270: 13850-13859Abstract Full Text Full Text PDF PubMed Scopus (113) Google Scholar, 67Schroeder S.C. Wang C.K. Weil P.A. J. Biol. Chem. 1994; 269: 28335-28346Abstract Full Text PDF PubMed Google Scholar). Whereas Rap1p did not bind to the Ad2 MLP (lane 3), TBP binding at all three sites was potently inhibited when Rap1p and TBP were added simultaneously (lane 4 versus 5). However, either pre-binding of TBP to DNA (lane 9) or TFIIA addition (lane 7) protected TBP from the negative action of Rap1p. Similar results were observed with RPS1A DNA, a yeast gene containing consensus (RAP1) and non-consensus (RAP1*) Rap1p binding sites (Fig. 1B, right, RPS1A). In this case, Rap1p both bound to RAP1 sites (lane 13) and blocked TBP-DNA binding to RPS1A TA-rich sequences (TA-I,-II, -III, -IV; lane 15). Again, TFIIA addition (lane 17) and TBP-DNA complex formation (lane 19) prevented Rap1p inhibition. The extent of inhibition of TBP-DNA binding by Rap1p was dependent on the concentration of both proteins, consistent with a direct, bimolecular Rap1p-TBP interaction (Fig. 1C, compare lanes 5-9, 10-14, and 15-19). Notably, TFIIB failed to block Rap1p from inhibiting TBP-TATA binding (Fig. 1D, compare lanes 20-22 with lanes 2-19 and 23-25). (Note that the apparent protection of DNA observed when both TBP and TFIIB are added to the footprinting assays is not reproducible (data not shown), and likely results from enhanced nuclease digestion of the probe when these two proteins are added simultaneously.) Together, these data suggested that Rap1p and TBP directly interacted, and that a specific domain(s) of TBP was targeted. Mapping Rap1p Domains That Inhibit TATA DNA Interaction through Direct TBP Binding—To map the TBP inhibitory region(s), we generated a family of Rap1p variants (Fig. 2, A and B) and assessed their ability to prevent TBP binding to Ad2 MLP (Fig. 2C, lanes 3-12) and RPS1A (Fig. 2C, lanes 26-35). The Rap1p DBD specifically (Fig. 2C, lanes 36-46 versus 13-23) inhibited TBP-DNA binding to the TATA regions of both DNAs (Fig. 2C, compare lanes 5, 6, 8, 10, 12, and 28, 29, 31, 33, 35 versus lanes 3, 4, 7, 9, 11, and 26, 27, 30, 32, 34). To test whether the Rap1p DBD directly bound TBP we performed GST-Rap1p pulldowns. We found that all GST-Rap1p variants that contained the Rap1p DBD specifically bound TBP (Fig. 2D, lanes 7, 9, 16, 21, 25 versus 12, 14, 18, 23, 27). Collectively these data demonstrated that the Rap1p DBD interacted with TBP directly, and suggested a molecular explanation for our observation that Rap1p blocked TBP-TATA binding. Mapping TBP Surfaces That Interact with Rap1p—To map the portions of TBP that interacted with Rap1p we performed pulldown competition assays to test whether well studied TBP-binding partners (TATA DNA, TFIIA, TFIIB, NC2 (Ncb1p and Ncb2p) and the Taf1p-TAND; Fig. 3A) inhibited TBP-Rap1p interaction. As expected from our previous work (58Banik 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, 59Bai Y. Perez G.M. Beechem J.M. Weil P.A. Mol. Cell. Biol. 1997; 17: 3081-3093Crossref PubMed Scopus (61) Google Scholar, 68Perez-Howard G.M. Weil P.A. Beechem J.M. Biochemistry. 1995; 34: 8005-8017Crossref PubMed Scopus (131) Google Scholar), all of the purified proteins (Fig. 3B) avidly bound TBP (Fig. 3C, lane 1 versus 2-6), indicating that these proteins were all highly active. When added along with TBP to GST-Rap1p binding reactions, neither TFIIB nor Ncb1p efficiently blocked TBP-Rap1p binding. By contrast, TFIIA, the NC2 Ncb2p subunit, and the Taf1p TAND all were able to block TBP-Rap1p complex formation quite efficiently (Fig. 3D, compare control lane 4, with lanes 7, 16, 19 versus lanes 10, 13). Similarly, double stranded WT (GGCTATAAAAGGGG), but not double stranded mutant (GGCTAAGAAAGGGG) Ad2 MLP TATA DNA prevented TBP-Rap1p complex formation (Fig. 3D, lane 21 versus 23). (We assume that neither Ncb1p nor TFIIB were GST-Rap1p-TBP associated due to a shorter half-life of these TBP complexes.) These data indicated that the DNA, NC2, TAND, and TFIIA interaction domains of TBP contributed importantly to Rap1p-TBP binding. Identification of TBP Residues That Interact with Rap1p—To more precisely map the Rap1p interaction domain we used a family of truncation and point mutant forms of TBP (Fig. 4A). To document the published activities of these TBP mutants (Fig. 4B), and to demonstrate that all variants had some definable biochemical activity and hence were not inactive in Rap1p binding due to the fact that they were, for example, just unfolded, we measured both dimerization (Fig. 4B, lanes 2-19, X-linked, anti-TBP IgG I-BLOT) and DNA binding, ±Rap1p and ±TFIIA (Fig. 4C). As reported (69Kou H. Irvin J.D. Huisinga K.L. Mitra M. Pugh B.F. Mol. Cell. Biol. 2003; 23: 3186-3201Crossref PubMed Scopus (14) Google Scholar, 70Kou H. Pugh B.F. J. Biol. Chem. 2004; 279: 20966-20973Abstract Full Text Full Text PDF PubMed Scopus (10) Google Scholar), these TBPs variably dimerized (Fig. 4B), and only TBP point mutant variants E186Q, Q68R, F177R, G180R, and N95R bound TATA. In every case when DNA binding was observed TATA binding was sensitive to Rap1p and protected by TFIIA (Fig. 4C). When tested via GST-TBP pulldown, all TBP" @default.
• W2086065602 created "2016-06-24" @default.
• W2086065602 creator A5005432791 @default.
• W2086065602 creator A5089471791 @default.
• W2086065602 date "2008-03-01" @default.
• W2086065602 modified "2023-10-16" @default.
• W2086065602 title "The Transcriptional Repressor Activator Protein Rap1p Is a Direct Regulator of TATA-binding Protein" @default.
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