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• W2004702466 abstract "The RNA polymerase II general transcription factor TFIID is a complex containing the TATA-binding protein (TBP) and associated factors (TAFs). We have used a mutant allele of the gene encoding yeast TAFII68/61p to analyze its functionin vivo. We provide biochemical and genetic evidence that the C-terminal α-helix of TAFII68/61p is required for its direct interaction with TBP, the stable incorporation of TBP into the TFIID complex, the integrity of the TFIID complex, and the transcription of most genes in vivo. This is the first evidence that a yeast TAFII other than TAFII145/130 interacts with TBP, and the implications of this on the interpretation of data obtained studying TAFIImutants in vivo are discussed. We have identified a high copy suppressor of the TAF68/61 mutation, TSG2, that has sequence similarity to a region of the SAGA subunit Ada1. We demonstrate that it directly interacts with TAFII68/61pin vitro, is a component of TFIID, is required for the stability of the complex in vivo, and is necessary for the transcription of many yeast genes. On the basis of these functions, we propose that Tsg2/TAFII48p is the histone 2A-like dimerization partner for the histone 2B-like TAFII68/61p in the yeast TFIID complex. The RNA polymerase II general transcription factor TFIID is a complex containing the TATA-binding protein (TBP) and associated factors (TAFs). We have used a mutant allele of the gene encoding yeast TAFII68/61p to analyze its functionin vivo. We provide biochemical and genetic evidence that the C-terminal α-helix of TAFII68/61p is required for its direct interaction with TBP, the stable incorporation of TBP into the TFIID complex, the integrity of the TFIID complex, and the transcription of most genes in vivo. This is the first evidence that a yeast TAFII other than TAFII145/130 interacts with TBP, and the implications of this on the interpretation of data obtained studying TAFIImutants in vivo are discussed. We have identified a high copy suppressor of the TAF68/61 mutation, TSG2, that has sequence similarity to a region of the SAGA subunit Ada1. We demonstrate that it directly interacts with TAFII68/61pin vitro, is a component of TFIID, is required for the stability of the complex in vivo, and is necessary for the transcription of many yeast genes. On the basis of these functions, we propose that Tsg2/TAFII48p is the histone 2A-like dimerization partner for the histone 2B-like TAFII68/61p in the yeast TFIID complex. TATA-binding protein RNA polymerase II-specific TBP-associated factors SPT-ADA-GCNS histone acetyltransferase complex temperature-sensitive minus yeast extract-peptone medium polyacrylamide gel electrophoresis glutathioneS-transferase hemagglutinin Initiation of RNA polymerase II transcription is regulated through the concerted actions of general transcription factors, which bind near the polymerase start site, and specific transcriptional activator proteins, which bind at more distant sites (1.Hampsey M. Microbiol. Mol. Biol. Rev. 1998; 62: 465-503Crossref PubMed Google Scholar, 2.Tjian R. Maniatis T. Cell. 1994; 77: 5-8Abstract Full Text PDF PubMed Scopus (955) Google Scholar, 3.Zawel L. Reinberg D. Annu. Rev. Biochem. 1995; 64: 533-561Crossref PubMed Scopus (391) Google Scholar). Sequence-specific gene regulatory proteins are thought to target multiple components of the general transcriptional machinery by direct protein-protein interactions or through coactivator proteins (3.Zawel L. Reinberg D. Annu. Rev. Biochem. 1995; 64: 533-561Crossref PubMed Scopus (391) Google Scholar, 4.Burley S.K. Roeder R.G. Annu. Rev. Biochem. 1996; 65: 769-799Crossref PubMed Scopus (628) Google Scholar). Much focus has been placed on TFIID, which is composed of the TATA-binding protein (TBP)1 and a collection of tightly associated factors commonly referred to as TAFIIs (4.Burley S.K. Roeder R.G. Annu. Rev. Biochem. 1996; 65: 769-799Crossref PubMed Scopus (628) Google Scholar, 5.Verrijzer C.P. Tjian R. Trends Biochem. Sci. 1996; 21: 338-342Crossref PubMed Scopus (319) Google Scholar). A plethora of biochemical analyses of mammalian andDrosophila TFIID indicates that TAFIIs play a crucial role in promoter recognition and may be a target of some activator proteins (4.Burley S.K. Roeder R.G. Annu. Rev. Biochem. 1996; 65: 769-799Crossref PubMed Scopus (628) Google Scholar, 5.Verrijzer C.P. Tjian R. Trends Biochem. Sci. 1996; 21: 338-342Crossref PubMed Scopus (319) Google Scholar). The identification of yeast TAFIIs (6.Reese J.C. Apone L. Walker S.S. Griffin L.A. Green M.R. Nature. 1994; 371: 523-527Crossref PubMed Scopus (148) Google Scholar, 7.Poon D. Bai Y. Campbell A.M. Bjorklund S. Kim Y.J. Zhou S. Kornberg R.D. Weil P.A. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 8224-8228Crossref PubMed Scopus (118) Google Scholar) and the subsequent analysis of mutants in vivo (8.Apone L.M. Virbasius C.A. Reese J.C. Green M.R. Genes Dev. 1996; 10: 2368-2380Crossref PubMed Scopus (130) Google Scholar, 9.Moqtaderi Z. Bai Y. Poon D. Weil P.A. Struhl K. Nature. 1996; 383: 188-191Crossref PubMed Scopus (251) Google Scholar, 10.Walker S.S. Reese J.C. Apone L.M. Green M.R. Nature. 1996; 383: 185-188Crossref PubMed Scopus (213) Google Scholar) altered our understanding of the role of TAFIIs in the regulation of transcription. Despite forming a discrete complex, mutation of different TAFII genes can have very different effects on the structure and function of the TFIID complex (11.Hahn S. Cell. 1998; 95: 579-582Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar). Mutation or depletion of seven different yeast TAFIIs failed to result in global changes in gene expression (8.Apone L.M. Virbasius C.A. Reese J.C. Green M.R. Genes Dev. 1996; 10: 2368-2380Crossref PubMed Scopus (130) Google Scholar, 9.Moqtaderi Z. Bai Y. Poon D. Weil P.A. Struhl K. Nature. 1996; 383: 188-191Crossref PubMed Scopus (251) Google Scholar, 10.Walker S.S. Reese J.C. Apone L.M. Green M.R. Nature. 1996; 383: 185-188Crossref PubMed Scopus (213) Google Scholar, 12.Holstege F.C.P. Jennings E.G. Wyrick J.J. Lee T.I Hengartner C.J. Green M.R. Golub T.R. Lander E.S. Young R.A. Cell. 1998; 95: 717-728Abstract Full Text Full Text PDF PubMed Scopus (1598) Google Scholar). Most notably, mutation or depletion of TAFII145p, the dTAFII250 homologue, affects only a small portion of the genome (9.Moqtaderi Z. Bai Y. Poon D. Weil P.A. Struhl K. Nature. 1996; 383: 188-191Crossref PubMed Scopus (251) Google Scholar, 10.Walker S.S. Reese J.C. Apone L.M. Green M.R. Nature. 1996; 383: 185-188Crossref PubMed Scopus (213) Google Scholar, 12.Holstege F.C.P. Jennings E.G. Wyrick J.J. Lee T.I Hengartner C.J. Green M.R. Golub T.R. Lander E.S. Young R.A. Cell. 1998; 95: 717-728Abstract Full Text Full Text PDF PubMed Scopus (1598) Google Scholar, 13.Walker S.S. Shen W.C. Reese J.C. Apone L.M. Green M.R. Cell. 1997; 90: 607-614Abstract Full Text Full Text PDF PubMed Scopus (129) Google Scholar, 14.Shen W.C. Green M.R. Cell. 1997; 90: 615-624Abstract Full Text Full Text PDF PubMed Scopus (167) Google Scholar). In contrast, inactivation of temperature-sensitive mutants of yeast TAF68/61,TAF60, TAF40, TAF25, andTAF17 resulted in much broader transcriptional phenotypes (15.Apone L.M. Virbasius C.A. Holstege F.C.P. Wamg J Young R.A. Green M.R. Mol. Cell. 1998; 2: 653-661Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar, 16.Michel B. Komarnitsky P. Buratowski S. Mol. Cell. 1998; 2: 663-673Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar, 17.Moqtaderi Z. Keaveney M. Struhl K. Mol. Cell. 1998; 2: 675-682Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar, 18.Krishnamurthy Jackson B.M. Rhee E. Hinnebusch A.G. Mol. Cell. 1998; 2: 683-692Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 19.Komarnitsky P.B. Michel B. Buratowski S. Genes Dev. 1999; 13: 2484-2489Crossref PubMed Scopus (53) Google Scholar, 20.Sanders S.L. Klebnaow E.R. Weil P.A. J. Biol. Chem. 1999; 274: 18847-18850Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar). The interpretation of the results obtained using TAFII mutants has been complicated by the presence of certain TAFIIs in the SAGA histone acetyltransferase complex (21.Grant P.A. Schieltz D. Pray-Grant M.G. Steger D.J. Reese J.C. Yates J. Workman J.L. Cell. 1998; 94: 45-53Abstract Full Text Full Text PDF PubMed Scopus (386) Google Scholar, 22.Ogryzko V.V. Kotani T. Zhang X. Schlitz R.L. Howard T. Yang X.J. Howard B.H. Qin J. Nakatani Y. Cell. 1998; 94: 35-44Abstract Full Text Full Text PDF PubMed Scopus (467) Google Scholar). It has been proposed that the broad transcriptional phenotypes of some TAFII mutants may result from their presence in both TFIID and the SAGA histone acetyltransferase complex (17.Moqtaderi Z. Keaveney M. Struhl K. Mol. Cell. 1998; 2: 675-682Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar). However, although presence of some TAFIIs in both TFIID and SAGA may contribute to broader transcriptional phenotypes, it cannot solely explain these effects. Mutations in TAF40, whose translation product is believed to be part of TFIID but not SAGA, cause a broad transcriptional phenotype (19.Komarnitsky P.B. Michel B. Buratowski S. Genes Dev. 1999; 13: 2484-2489Crossref PubMed Scopus (53) Google Scholar). There is a strong correlation between the severity of the effects of TAFII mutations on the integrity of the TFIID complexin vivo and the fraction of the genome affected by their inactivation. All TAFIIs whose inactivation leads to decreases in the expression of a large percentage of the genome cause a concurrent reduction of additional TAF subunits and TBP in vivo. In particular, the TAFII genes with similarities to the core histones are important for the structure of TFIID in vivo. In contrast, mutation or depletion of the two largest yeast TAFIIs, TSM1p or TAFII145p, has relatively minor effects on TFIID structure (9.Moqtaderi Z. Bai Y. Poon D. Weil P.A. Struhl K. Nature. 1996; 383: 188-191Crossref PubMed Scopus (251) Google Scholar, 10.Walker S.S. Reese J.C. Apone L.M. Green M.R. Nature. 1996; 383: 185-188Crossref PubMed Scopus (213) Google Scholar). This is surprising considering that yTAFII145p (dTAFII250) is the subunit that binds to TBP with highest affinity (6.Reese J.C. Apone L. Walker S.S. Griffin L.A. Green M.R. Nature. 1994; 371: 523-527Crossref PubMed Scopus (148) Google Scholar, 22.Ogryzko V.V. Kotani T. Zhang X. Schlitz R.L. Howard T. Yang X.J. Howard B.H. Qin J. Nakatani Y. Cell. 1998; 94: 35-44Abstract Full Text Full Text PDF PubMed Scopus (467) Google Scholar, 23.Kokubo T. Swanson M.J. Nishikawa J-I Hinnebusch A.G. Nakatani Y. Mol. Cell. Biol. 1998; 18: 1003-1012Crossref PubMed Scopus (102) Google Scholar, 24.Bai Y. Perez G.M. Beechem J.M. Weil P.A. Mol. Cell. Biol. 1997; 17: 3081-3093Crossref PubMed Scopus (61) Google Scholar), and dTAFII250 is absolutely essential to form a functional TFIID complex in biochemical reconstitution experiments (25.Chen J.L. Attardi L.D. Verrijzer C.P. Yokomori K. Tjian R. Cell. 1994; 79: 93-105Abstract Full Text PDF PubMed Scopus (328) Google Scholar). A logical explanation for these observations is that additional TFIID subunits bind to TBP in the absence of TAFII145p, and that the histone-like TAFIIs are more important for TFIID structure. Structural and biochemical studies on metazoan TAFIIs revealed that three TAFIIs adopt a structure similar to the histone fold of the core histones and are capable of forming dimer pairs (26.Hoffmann A. Chiang C.M. Oelgeschlager T. Xie X. Burley S.K. Nakatani Y. Roeder R.G. Nature. 1996; 380: 356-359Crossref PubMed Scopus (159) Google Scholar, 27.Xie X. Kokubo T. Cohen S.L. Mirza U.A. Hoffmann A. Chait B.T. Roeder R.G. Nakatani Y. Burley S.K. Nature. 1996; 380: 316-322Crossref PubMed Scopus (228) Google Scholar, 28.Birck C. Poch O. Romier C. Ruff M. Mengus G. Lavigne A.-C. Davidson I. Moras D. Cell. 1998; 94: 239-249Abstract Full Text Full Text PDF PubMed Scopus (116) Google Scholar). The structure of the dTAFII60/dTAFII40 (yTAFII60/yTAFII17) dimer resembles that of histone H3/H4 in the nucleosome, and the primary sequence of hTAFII20 (yTAFII68/61) predicts its structure resembles histone H2B. The histone 2A-like dimerization partner of hTAFII20 (yTAFII68/61) in the hTFIID complex has been recently identified as hTAFII135 (29.Gangloff Y.-G. Werten S. Romier C. Carre L. Poch O. Moras D. Davidson I. Mol. Cell. Biol. 2000; 20: 340-351Crossref PubMed Scopus (86) Google Scholar). These same studies identified the SAGA component Ada1, as the H2A partner of yTAFII68/61p in the SAGA complex. Unfortunately, there is no yeast protein that shows extensive sequence homology to hTAFII135, leaving the dimerization partner of TAFII68/61p in the yTFIID complex unidentified. The analysis of yeast TAFII68/61 contained in this report has addressed two important issues regarding the function of this gene and its role in the yTFIID complex. First, we have identified the putative C-terminal α-helix of TAFII68/61p as important for the interaction of TBP with the yTFIID complex, indicating that TAFII145p is not the only subunit capable of binding to TBP. Second, we have isolated a gene, TSG2/MPT1, as a suppressor of a TAF68/61 mutation and demonstrate that it can stabilize the mutant TFIID complex in vivo. Finally, we have identified Tsg2/TAFII48p as a bona fideTFIID subunit and provide genetic, molecular, and biochemical evidence that it is the dimerization partner for TAFII68/61p in the TFIID complex. pRS414-TAF68/61 was mutagenized in vitro for 60–120 min at 70 °C using hydroxylamine, purified and transformed directly into YJR20 (Mat a;Δtaf68:hisg; ade2–11; his3–11, 15; leu2–3, 112; ura3–1; trp1–1; can1–100; [RS416-TAF68/61]). Tryptophan prototrophs were replica plated onto 5-fluoroorotic acid-containing media at 23 °C and 37 °C, and mutants that supported growth at 23 °C, but not 37 °C, were isolated for further analysis. The ts− phenotypes were verified to be plasmid-linked by recovery of the plasmids, re-transformation into YJR20, and rescreening at the indicated temperatures. Mutants that ceased growth at ≤2 doubling times were analyzed further. The strain YJR21–9 (taf68–9) was transformed with a Yep13-based high copy genomic library obtained from the ATTC. Approximately 40,000 transformants were plated at 23 °C for 16 h and than transferred to 37 °C for 2–5 days. Colonies were isolated, and the plasmids were recovered from the cells and reintroduced into YJR21–9. Plasmids that conferred growth at 37 °C were sequenced, and the open reading frame conferring the suppression phenotype was identified by subcloning into pRS426 and rescreening in YJR21–9. The plasmid pGAL1-TSG2/TAF48 was constructed by cloning the polymerase chain reaction-amplified coding sequence ofTSG2/TAF48 into the vector pSW107 (10.Walker S.S. Reese J.C. Apone L.M. Green M.R. Nature. 1996; 383: 185-188Crossref PubMed Scopus (213) Google Scholar) and integrated at theTRP3 locus of the strain BY4705 (Mat α; ade2Δ::hisg; his3Δ200; leu2Δ0; lys2Δ0; met15Δ0; trp1Δ63; ura3Δ0). After verification, the chromosomal TSG2/TAF48 gene was deleted by homologous recombination using a polymerase chain reaction-based strategy resulting in the strain YJR475 (Mat α; ade2Δ::hisg; his3Δ200; leu2Δ0; lys2Δ0; met15Δ0; trp1Δ63; ura3Δ0;Δtsg2::LEU2; trp3::GAL1p-HA-TSG2::HIS3). Cultures of YJR475 were grown to an optical density of 1.0 in YP plus galactose, collected by centrifugation, washed once in YP, and grown in YP plus dextrose for the times indicated in the Fig. legends. Cells were grown to midlog phase in YP plus dextrose (unless indicated otherwise) and shifted to a 37 °C shaking water bath. Aliquots were taken from the culture prior to temperature shift and at 0.5, 1, 2, and 4 h thereafter. After centrifugation, cells were washed in ice-cold STE (Tris-EDTA plus 100 mmNaCl) and separated into two parts for the isolation of RNA and protein extract preparation. RNA was prepared (8.Apone L.M. Virbasius C.A. Reese J.C. Green M.R. Genes Dev. 1996; 10: 2368-2380Crossref PubMed Scopus (130) Google Scholar, 10.Walker S.S. Reese J.C. Apone L.M. Green M.R. Nature. 1996; 383: 185-188Crossref PubMed Scopus (213) Google Scholar) and analyzed by S1 nuclease protection (30.Cormack B.P. Struhl K. Cell. 1992; 69: 685-696Abstract Full Text PDF PubMed Scopus (277) Google Scholar) or Northern blotting. Protein extracts were prepared by glass-bead disruption in 50 mm Tris-HCl, pH 8.0, 5 mm EDTA, 0.7 m NaCl, 0.1% Triton X-100, 10% glycerol, 5 mm dithiothreitol, 0.1 mmsodium orthovanadate, 25 mm sodium fluoride, and protease inhibitors (6.Reese J.C. Apone L. Walker S.S. Griffin L.A. Green M.R. Nature. 1994; 371: 523-527Crossref PubMed Scopus (148) Google Scholar). We find that the addition of NaCl to >0.6m significantly aids in the extraction of TAFs and other transcription factors. 20 μg of whole cell extract protein was fractionated on SDS-PAGE gels and transferred to nitrocellulose in buffers containing and lacking SDS, depending on the TAFs to be analyzed. Antibodies and Western blotting were described (10.Walker S.S. Reese J.C. Apone L.M. Green M.R. Nature. 1996; 383: 185-188Crossref PubMed Scopus (213) Google Scholar). Mid-scale extracts were prepared from 1-liter cultures of cells grown at the permissive temperature as follows. Cells were collected by centrifugation and washed in ice-cold STE plus 1 mmbenzamidine HCl and 0.2 mm phenylmethylsulfonyl fluoride and frozen. One cell pellet volume of buffer T containing 0.3m potassium acetate plus 0.1% Nonidet P-40 and protease inhibitors (6.Reese J.C. Apone L. Walker S.S. Griffin L.A. Green M.R. Nature. 1994; 371: 523-527Crossref PubMed Scopus (148) Google Scholar) and 1 volume of glass beads were added, and the cells were broken by vortex mixing for 6 cycles of mixing and cooling. The beads and debris were separated by low speed centrifugation, and the supernatant was further clarified by centrifugation twice for 30 min at 30,000 × g. TFIID was immunoprecipitated from 1 ml of extracts diluted to 2–3 mg/ml protein with 0.3 m buffer T as follows. Antiserum was added for 2–4 h on ice, followed by an overnight incubation with protein A-agarose beads (Repligen). Protein A beads were collected by low speed centrifugation and washed three times for 10 min each in buffer T containing the concentrations of potassium acetate indicated in the figure legends. After a brief wash in buffer T containing 0.1 m potassium acetate, proteins were eluted in SDS-PAGE loading buffer. The region of TAF68and taf68–9 encoding the conserved C-terminal portion of the protein was amplified by polymerase chain reaction and cloned into the BamHI and EcoRI sites of pGEX-1. GlutathioneS-transferase (GST) proteins were produced in XA90 cells and purified on glutathione-agarose beads, and Bradford assays and Coomassie Blue-stained SDS-PAGE gels monitored protein concentrations. Equal amounts of GST fusion protein was bound to the beads. Yeast TBP and Tsg2/TAF48p was in vitro translated from pet15-yTBP (31.Hoffmann A. Roeder R.G. J. Biol. Chem. 1996; 271: 18194-18202Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar) and bluescript-TSG2/TAF48, respectively, using a wheat germ transcription and translation (TnT) kit from Promega. Three micrograms of GST fusion protein bound to 5 μl of glutathione beads were incubated with 20 μl of wheat germ extract and 80 μl of binding buffer (20 HEPES-KOH, pH 7.5, 150 mm potassium acetate, 1 mm EDTA, 1 mm dithiothreitol, 10% (v/v) glycerol, and 0.01% Nonidet P-40) at 4 °C for 60 min with agitation. Afterward, 200 μl of ice-cold binding buffer was added, and the beads were collected by low speed centrifugation. The beads were washed 4 times for 5 min each with 300 μl of binding buffer and finally eluted with SDS-PAGE loading buffer. The bound proteins were separated by SDS-PAGE, stained with Coomassie Blue, treated with En3Hance (NEN Life Science Products), dried, and exposed to film. In vitro random chemical mutagenesis and plasmid shuffling was used to isolate three TAF68/61 mutants that could not support growth at 37 °C (Fig.1 A). The sequence of the mutants was determined, and it was found that all three alleles contained a single nucleotide change at nucleotide 1456 (ATG = +1) resulting in the change from a tryptophan residue to a stop codon at amino acid 486. The expressed protein is truncated for the last 54 amino acids (Fig. 1 B). It is interesting to note that the only tight ts− mutants we isolated out of a total 20,000 colonies screened contained the same mutation. Moreover, two ts− alleles ofTAF68/61 described in another report also contained truncations at and near this region (16.Michel B. Komarnitsky P. Buratowski S. Mol. Cell. 1998; 2: 663-673Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). Truncation at residue 486 removed the C-terminal α-helix (Fig. 1 B) but retained the three α-helices comprising the histone fold (32.Burley S.K. Xie X. Clark K.L. Shu F. Curr. Opin. Struct. Biol. 1997; 7: 94-102Crossref PubMed Scopus (37) Google Scholar). Inactivation of three different yeast TAFII mutants results in a specific cell cycle arrest phenotype (8.Apone L.M. Virbasius C.A. Reese J.C. Green M.R. Genes Dev. 1996; 10: 2368-2380Crossref PubMed Scopus (130) Google Scholar, 10.Walker S.S. Reese J.C. Apone L.M. Green M.R. Nature. 1996; 383: 185-188Crossref PubMed Scopus (213) Google Scholar); we therefore analyzed the cell cycle phenotypes of the taf68–9 mutant following growth at the restrictive temperature. We found that shifting the taf68–9 mutant to the restrictive temperature caused a rapid cessation of growth with no specific cell cycle phenotypes (not shown). A previous study showed that inactivation of temperature-sensitive mutants ofTAF68/61 resulted in the reduced transcription of a large number of yeast genes (16.Michel B. Komarnitsky P. Buratowski S. Mol. Cell. 1998; 2: 663-673Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar). We therefore examined the effects oftaf68–9 inactivation on the transcription of yeast genes to verify the phenotype in our strain. A wild type strain, thetaf68–9 mutant, and as a control, a temperature-sensitive RNA polymerase II mutant (rpb1–1), were transferred to the restrictive temperature, and RNA was isolated at various times. The mRNA levels of seven representative genes were analyzed by S1 nuclease protection. Within 1 h of temperature shift, we found significant reductions in the mRNA levels of a number of RNA polymerase II-transcribed genes (Fig. 2 A and data not shown), but only minor effects were observed on the levels ofSPT15 and DED1 mRNA. The expression of an RNA polymerase III-transcribed gene (tRNA) was only mildly affected at later time points (Fig. 2 A). We next examined the effect of the inactivation of taf68–9 on the expression of a number of highly induced genes. The wild type and taf68–9 mutant was preincubated at 37 °C for 1 h and then treated under the inducing conditions described in the figure legends. Growth of thetaf68–9 mutant at 37 °C prevented the induction of theRNR2 gene by DNA damage and the derepression of theSUC2 gene (Fig. 2 B). Moreover, we found that the uninduced levels of SUC2 and RNR2 were reduced in the mutant. However, inactivation of taf68–9 did not affect the induction of all genes tested. Induction of the SSA4,CUP1, and CTT1 were not significantly reduced by the inactivation of the taf68–9 mutant but were strongly affected by mutations in the large subunit of RNA polymerase II (Fig.2 B). Incubation of certain TAFII mutants at the restrictive temperature results in the disruption of the TFIID complex and the degradation of TAFII subunits in vivo (10.Walker S.S. Reese J.C. Apone L.M. Green M.R. Nature. 1996; 383: 185-188Crossref PubMed Scopus (213) Google Scholar, 16.Michel B. Komarnitsky P. Buratowski S. Mol. Cell. 1998; 2: 663-673Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar,19.Komarnitsky P.B. Michel B. Buratowski S. Genes Dev. 1999; 13: 2484-2489Crossref PubMed Scopus (53) Google Scholar, 20.Sanders S.L. Klebnaow E.R. Weil P.A. J. Biol. Chem. 1999; 274: 18847-18850Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar); therefore, we analyzed the effects of shifting thetaf68–9 mutant to the restrictive temperature on the steady state levels of TAFII protein. We found that incubation of the mutant at 37 °C caused a rapid reduction in the levels of TBP, TAFII145p, and TAFII47p and, to a lesser degree, TAFIIs 90p, 68p, 60p, 25p, and 17p (Fig.2 C and data not shown). In contrast, Sua7p, a nuclear protein not associated with TFIID or SAGA, was not affected. It appears that the TAFIIs not shared between TFIID and the SAGA complex (21.Grant P.A. Schieltz D. Pray-Grant M.G. Steger D.J. Reese J.C. Yates J. Workman J.L. Cell. 1998; 94: 45-53Abstract Full Text Full Text PDF PubMed Scopus (386) Google Scholar) are less stable in the mutant. Shifting a TBP (tbp1–1; Fig. 2 C) or a RNA polymerase II ts− mutant (not shown) to the restrictive temperature failed to cause similar reductions in TAFII protein levels, indicating that the reductions in TBP and TAFII protein in the taf68–9 mutant is largely at the level of protein turnover. Interestingly, growth of the TBP mutant at 37 °C resulted in the rapid turnover of TBP but not other subunits in the TFIID complex, indicating that TBP is not required for the stability of TAFIIs. Our results above demonstrate that inactivation of temperature-sensitive mutants ofTAF68/61 causes a loss of TBP protein within the cell that is faster and more severe than what is observed in TAF145mutants (10.Walker S.S. Reese J.C. Apone L.M. Green M.R. Nature. 1996; 383: 185-188Crossref PubMed Scopus (213) Google Scholar). This result is surprising in light of the fact that TAFII145p was identified as the single subunit of TFIID that binds to TBP with high affinity by Far Western blotting (6.Reese J.C. Apone L. Walker S.S. Griffin L.A. Green M.R. Nature. 1994; 371: 523-527Crossref PubMed Scopus (148) Google Scholar). A logical explanation for these findings is that in the absence of TAFII145p another TFIID subunit binds to TBP and protects it from degradation. The human homologue of TAFII68/61p (hTAFII20) can bind to TBP in vitro (31.Hoffmann A. Roeder R.G. J. Biol. Chem. 1996; 271: 18194-18202Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar), and it is likely that TAFII68/61p has this function. To test this hypothesis, we expressed the conserved C-terminal portion (amino acids 330–539) of TAFII68/61p as a GST derivative and used it in pull down assays to identify an interaction with in vitrotranslated TBP. This region alone retains all the essential functions of the full-length TAFII68/61protein because it can fully complement a TAF68/61 gene disruption (33.Moqtaderi Z. Yale J.D. Struhl K. Buratowski S. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 14654-14658Crossref PubMed Scopus (84) Google Scholar). A GST derivative containing the activation domain of the herpesvirus protein VP16 was used as a positive control in our experiments. TBP was capable of interacting with the C terminus of TAFII68/61p; moreover, it bound to TBP more strongly than GST-VP16 (Fig.3 B). However, when the assay was carried out using a GST derivative corresponding to thetaf68–9 mutation, GST-taf68–9C (amino acids 330–485), we found the mutant was defective for its ability to interact with TBPin vitro (Fig. 3 B). The binding of TBP to the N terminus of TAFII145p and dTAFII230 has been extensively characterized (6.Reese J.C. Apone L. Walker S.S. Griffin L.A. Green M.R. Nature. 1994; 371: 523-527Crossref PubMed Scopus (148) Google Scholar, 22.Ogryzko V.V. Kotani T. Zhang X. Schlitz R.L. Howard T. Yang X.J. Howard B.H. Qin J. Nakatani Y. Cell. 1998; 94: 35-44Abstract Full Text Full Text PDF PubMed Scopus (467) Google Scholar, 23.Kokubo T. Swanson M.J. Nishikawa J-I Hinnebusch A.G. Nakatani Y. Mol. Cell. Biol. 1998; 18: 1003-1012Crossref PubMed Scopus (102) Google Scholar,34.Liu D. Ishima R. Tong K. Bagby S. Kokubo T. Munhandiram D.R. Kay L.E. Nakatani Y Ikura M. Cell. 1998; 94: 573-583Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar). To address the significance of the TBP-TAFII68/61 interaction, we compared the TBP binding ability of GST-TAFII68C versus GST-TAFII145N (amino acids 5–230) under identical conditions. The results contained in Fig. 3 C clearly show that TBP interacted equally well with the C terminus of TAFII68/61p and the N terminus of TAFII145p. Our TBP binding studies demonstrate that the C-terminal α-helix (485–539) of TAFII68/61p is required for its ability to bind to TBP and that this interaction is comparable with that of the well characterized TAFII145p-TBP interaction. We next sought to determine if the C terminus of TAFII68/61p is important for the interaction of TBP with the TFIID complex by comparing the ability of TFIID to co-immunoprecipitate with TBP in extracts of wild type and mutant cells. For this experiment, extracts were prepared from cells grown at the permissive temperature because incubation at 37 °C would result in the depletion of TAFs and ambiguous results. We failed to detect any differences in the co-immunoprecipitation of TFIID in extracts from wild type and mutant cells under our standard medium salt conditions (Fig. 3 D and data not shown). Since TAFII145p is present in these complexes, it is likely that its interaction with TBP is obscuring the detection of any differences between the wild type and mutant TFIID complexes that would otherwise be apparent in vivo. Therefore, we performed our analysis under conditions that disrupt the TBP-TAFII145Np interaction and accentuate the contributions of other TBP-TAF interactions. The TAFII145Np-TBP interaction is sensitive to pot" @default.
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