DAVID POON*, Yu BAI*, ALLYSON M. CAMPBELL*, STEFAN I3JORKLUNDt, ...... Mallet, L., Demolis, N., Bussereau, F. & Jacquet, M. (1994) Yeast 10,. 819-831.
Proc. Natl. Acad. Sci. USA Vol. 92, pp. 8224-8228, August 1995 Biochemistry
Identification and characterization of a TFIID-like multiprotein complex from Saccharomyces cerevisiae DAVID POON*, Yu BAI*, ALLYSON M. CAMPBELL*, STEFAN I3JORKLUNDt, YOUNG-JOON KIMt, SHARLEEN ZHOUt, ROGER D. KORNBERGt, AND P. ANTHONY WEIL*§ *Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, TN 37232-0615; tDepartment of Cell Biology, Stanford University School of Medicine, Stanford, CA 94305; and tHoward Hughes Medical Institute, Department of Molecular and Cellular Biology,
University of Calitornia, Berkeley, CA 94720 Communicated by Stanley Cohen, Vanderbilt University, Nashville, TN, April 20, 1995
spite the genetic isolation of yeast genes which encode factors involved in mediating transcriptional activation (17, 18). Clearly if the mechanisms of transcriptional regulation are truly conserved between unicellular and multicellular organisms, then a TFIID-like TBP-TAFI multiprotein complex containing coactivator activity should also exist in yeast. We previously showed that yeast TBP (yTBP), which is required for transcription by all three RNA polymerases (19, 20), is associated with at least nine distinct proteins ranging in size from "170 kDa to 25 kDa (21-23). We demonstrated that all of the components of the RNA polymerase III transcription factor TFIIIB, a known TBP-TAF complex, which includes the TAF,1170 protein Brflp (also known as Tds4p and Pcf4p; refs. 24-26), were present in this TAF fraction. These data indicate that at least two of the TAFs of this protein fraction are RNA polymerase III specific. On the basis of these results, we therefore hypothesized that this TAF fraction might also contain RNA polymerase II-specific TAFs, particularly, a yeast multiprotein complex-i.e., TBP-TAF11s-comparable to metazoan TFIID. In this report, we describe the cloning and sequence of the genes encoding several of the yeast TAFs (yTAFs) in this TAF protein fraction and demonstrate that these yTAFs are, in fact, associated with TBP.'g We also show that this TBP-TAFI, complex has the biochemical and genetic hallmarks of metazoan TFIIDs. Finally, we document that the genes encoding yeast TAF11s are essential for yeast cell viability and discuss the potential interrelationships of this yeast T3P-TAFI, complex and yeast RNA polymerase II holoenzyme (27, 28) in transcriptional regulation. While this manuscript was in preparation, a report appeared by Reese et al. (29) that described a TBP-TAFI complex which exhibits similar features to those described herein.
ABSTRACT Although the mechanisms of transcriptional regulation by RNA polymerase II are apparently highly conserved from yeast to man, the identification of a yeast TATAbinding protein (TBP)-TBP-associated factor (TAFII) complex comparable to the metazoan TMIID component of the basal transcriptional machinery has remained elusive. Here, we report the isolation of a yeast TBP-TAF]g complex which can mediate transcriptional activation by GAL4-VP16 in a highly purified yeast in vitro transcription system. We have cloned and sequenced the genes encoding four of the multiple yeast TAFI proteins comprising the TBP-TAFH multisubunit complex and find that they are similar at the amino acid level to both human and Drosophila TFIID subunits. Using epitopetagging and immunoprecipitation experiments, we demon. strate that these genes encode bona fide TAF proteins and show that the yeast TBP-TAFI, complex is minimally composed of TBP and seven distinct yTAFIn proteins ranging in size from Mr = 150,000 to Mr = 25,000. In addition, by constructing null alleles of the cloned TAF-encoding genes, we show that normal function of the TAF-encoding genes is essential for yeast cell viability. The regulation of transcription of mRNA-encoding eukaryotic genes is a complicated process involving the modulation of chromatin structure, activities of upstream activators and repressors, and the concerted action of multiple components of the basal transcription machinery, including RNA polymerase It itself (1, 2). It is thought that the interaction of the TATA-binding protein (TBP), with the TATA-box promoter element is the first step in the formation of the RNA polymerase II preinitiation complex (PIC), and numerous studies have shown that PIC formation is subject to modulation by a variety of transcriptional regulators. However, the mechanisms by which these factors exert their effects are not yet fully understood. In metazoan systems, one basal factor that has been shown to be directly involved in mediating activation by upstream activators is the transcription factor TFIID, which is composed of TBP and TBP-associated factors (TAF11s). Human and Drosophila TFIID complexes each contain at least eight TAF11s, and the genes encoding a number of these have been cloned and sequenced (3-15). Several TAFI1s have been shown to interact directly with the activation domains of known transcriptional activator proteins (12-16), and these interactions are thought to be integral in some way to the transactivation process. Although the mechanisms of transcriptional regulation are thought to be conserved from yeast to man, when we initiated our studies, a TFIID-like TBP-TAF1 multisubunit complex similar to human and Drosophila TFIIDs had not been directly biochemically identified from Saccharomyces cerevisiae, de-
MATERIALS AND METHODS Yeast Strains. YPH252 (30) is our standard wild-type yeast strain; gene disruptions were performed in diploid yeast strain SEY6210.5 (31). Protein extracts used for preparative yTAF protein purification by using anti-TBP IgG affinity chromatography were prepared from BJ5457 cells (21); hemagglutinin (HA)-tagged TBP-TAFj1 protein complexes were prepared from haploid yeast strains derived from sporulated SEY6210.5-derived clones. yTAFia Purification and Protein Sequencing. Yeast TAFI, proteins were preparatively purified as detailed (21, 22) and sequenced (32), and the sequence information was analyzed Abbreviations: TBP, TATA-binding protein; TAF, TBP-associated factor; yTBP, yeast TBP; yTAF, dTAF, and hTAF, yeast, Drosophila, and human TAF, respectively; Ab, antibody; mAb, monoclonal Ab; WCE, whole-cell extract; HA, hemagglutinin. §To whom reprint requests should be addressed. 1The sequences reported in this paper have been deposited in the GenBank data base (accession nos. L40145).
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
8224
Proc. Natl. Acad. Sci. USA 92 (1995)
B'iochemistry: Poon et aL via BLAST searches (33). The peptide sequences were as follows: yTAF11150, (aa 165-174) KTTPGFQESV and (aa 991-1000) KQFLLDILVY; yTAF,1130, (aa 391-407) SLIEDVAED)WQWDEDMI, (aa ;54-580) ESFSTSQDLTIGDTAPVYLMEYSEQTP, and (aa 789-820) SLITPEQISQVESMSQGLQFQEDNEAYNFDSK; yTAF,190, (aa 22-63) NQRTNNAAGANSGQQPQQQSQGQSQQQGRSNGPFSASDLNRI and (aa 726-752) ATTEPSAEPPDEPFIGYLGDVTASINQD; yTAF1160, (aa 79-99) ALRVLNVEPLYGYYDGSEVNK, (aa 106-139) VNTSGGQSVYYLDEEEVDFDRLINEPLPQVPRLP, and (aa 236-243) ELQIYFNK. The numbers refer to amino acid position within the deduced protein sequences, with the N-terminal methionine given position 1. TAF Gene Cloning. Intrapeptide PCR was performed by using degenerate oligonucleotides and either yeast genomic DNA or total yeast cDNA library DNA as template (34, 35), and appropriate-length PCR product fragments were sequenced. This information was used to obtain full-length clones of all the yTAFII-encoding genes, which were sequenced by using standard dideoxynucleotide/chain termination methods. Details of the sequencing, cloning and construction, and verification of taf-null alleles are available on request. Construction of HA-Tagged yTAF11-Encoding Genes. Yeast expression plasmids encoding yTAF11150, yTAFii130, yTAFI190, yTAFi160, and yTBP or their triple HA-tagged variants (36) were constructed basically as described (22) by using standard methods for DNA manipulation. Our Tsmlpencoding plasmid was kindly supplied by J. Haber (Brandeis University, Waltham, MA). Immunological Methods. Analytical and preparative-scale immunoprecipitation and immunoblotting experiments were performed with yeast whole-cell extract (WCE) protein fractions as described (21, 22). Purification and elution of the HA-tagged TAF-containing complexes were achieved by using a solution of peptide (sequence GGYPYDVPt)YAGGYPYDVPDYAGGYPYDVPDYAGGYPYDVPDYAGG) in BA/ 300 (1 mg/ml) (22) containing 0.1% Nonidet P-40 and 100 ,zg of insulin per ml as carrier (HA epitope sequence underlined). In Vitro Transcription Assays. Transcription reactions were performed essentially as described (28, 37, 38). Reaction mixtures contained 50-100 ng of purified transcription factors yTFIIB, -E, -F, and -H; 100 ng of purified yeast core RNA polymerase II; and 100 ng each of template pSPGCN4CG (UASGCN4 driven) and pJJ470 (UASGAL4 driven), supplemented with 50 ng of recombinant yTBP and/or 100 ng of GAL4-VP16 and/or 50-100 ng of yTAFs.
RESULTS Yeast TBP-TAFI5 Complexes Display TFIID-Like Coactivator Activity. To test whether our yTAF preparation (a typical yTAF polypeptide profile is shown in Fig. 1A) contained TFIID-like coactivator activity, we used a highly purified yeast in vitro transcription system to see if this protein fraction could mediate an RNA polymerase II transactivation event. We used purified GAL4-VP16 activator protein and essentially homogenous preparations of yeast TFIIB, -E, -F, and -H and core RNA polymerase II (28) for our transcription reconstitution assays (37, 38). As shown in Fig. iB, the yTAF protein fraction was able to specifically mediate a modest but reproducible 2.5to 3-fold activation by GAL4-VP16 without affecting transcription from the control GCN4-driven template. This result suggests that, as we hypothesized, our yeast TBP-TAF fraction does contain coactivator activity similar to that of metazoan TFIID. Cloned Yeast TAF-Encoding Genes Show Marked Sequence Similarities to Metazoan TAF1Is. To characterize the yTAFjjs further, the genes encoding four of them were cloned and sequenced. Individual TAF proteins were isolated, and pep-
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FIG. 1. Subunit composition and transcriptional activity of yTAFIiTBP complex. (A) Silver-stained g6l of proteins present in the yTAF fraction. yTBP and associated factors were purified by anti-TBP IgG affinity chromatography and fractionated by SDS/PAGE. The nine polypeptides that consistently coimmunopurify with yTBP are shown, and those thought to make up the RNA polymerase II-specific complex are labeled as yTAFiis. Asterisks indicate polypeptides that are retained by a preimmune IgG-protein A affinity matrix. The abundant polypeptide species migrating with a mass of 70 kDa was previously thought (21) to be entirely Brflp, but immunoblotting experiments using anti-Brflp antibodies (ref. 24; kindly supplied by S. Hahn) suggest that although Brflp migrates at this position, the amount of Brflp is less than the strongly silver-stained 70-kDa species would indicate. Thus, the 70-kDa species has been designated p70. yTBP migrates slightly slower than yTAFII25 and is difficult to visualize on these silver-stained gels, as neither yTBP nor yTAFII25 stains well with silver. (B) Effect of the addition of the yTAFIi fraction upon VP16-mediated transactivation. In vitro transcription reactions contained purified yTFIIB, yTFIIE, yTFIIF, yTFIIH, yeast core RNA polymerase II, and, as indicated, recombinant yTBP (ryTBP), yTAFs, or GAL4-VP16. Arrows indicate specific transcripts produced from the GCN4- (upper) and GAL4- (lower) driven templates. Phosphorimaging quantitation trorh this gel analysis for the UASGCN4-directed template is 1737, 1926,3553, and 3394, for lanes 1-4, respectively, and for the UASGAL-directed template is 4096, 3844, 1917, and 11502, for lanes 1-4, respectively.
tides derived from yTAFII150, -130, -90, and -60 were sequenced (see Materials and Methods for sequences). A data
Biochemistry: Poon et aL
8226
Proc. Natl. Acad. Sci. USA 92 (1995)
base search using the derived amino acid sequence information and the BLAST search algorithms (33) revealed that yTAFI150 is encoded by the essential yeast gene TSM1 (39) and that yTAF11130 and yTAF,160 ar, encoded by previously uncharacterized genes. Initial data base searches failed to find a match to yTAF,190; however, after this gene was cloned and partially sequenced, we found that yTAF5190 is encoded by ORFYBR1410, a gene present on chromosome 11 (40). Fulllength clones of the genes encoding yTAF,1130, yTAF 190, and yTAFI,60 were obtained as detailed in Materials and Methods. The gene encoding yTAF11130 (designated TAF130 for TBPassociated factor 130 kDa) consists of an open reading frame of 1066 amino acids, which could encode a protein of Mr = 120,685. The gene encoding yTAF,190 (for consistency herein designated TAF90 instead of ORFYBR1410) consists of an open reading frame of 798 amino acids with a deduced Mr = 88,960. The gene encoding yTAF1160 (TAF60) consists of an open reading frame of 516 amino acids with a deduced Mr = 57,897. Comparisons of the amino acid sequences predicted from the TAF150 (TSM1), TAF130, TAF90, and TAF60 genes with metazoan TAF-encoding genes reveal striking sequence similarities between the yTAFs and human and Drosophila TFIID subunits, a result which lends strong support to the hypothesis that the cloned yTAF genes encode actual subunits of a yeast TFIID multiprotein complex. yTAF,1150/Tsmlp is the yeast homolog of Drosophila TAF11150 (dTAFI150), and these two proteins are -50% similar at the amino acid level (41). TSM1 was originally identified by the isolation of a temperaturesensitive mutant in a region of chromosome III located to the right of the MATlocus (39). At the amino acid level, yTAFI130 is similar to both human TAF11250 (hTAFI250)/CCG1 and to
dTAF,1230/250 (Fig. 2A). In the region of highest similarity, this yTAF is 78% similar to its metazoan counterpart (yTAFI130 amino acids 440-830; refs. 3, 4, and 6). Thus, at this level of analysis, it appears that the N-terminal 60% of the metazoan TAFI1250 is conserved within yTAFI130 sequences, a conclusion also reached by Reese et al. (29). On the basis of overall length and sequence similarity, yTAFI90 appears to be the yeast homolog of dTAF1180 (Fig. 2B). yTAF1190 is 62% similar to dTAF1180 at its C terminus (7). The deduced amino acid sequence of yTAFii90 also contains potential l3-transducin (WD40) repeats like its metazoan counterpart. These repeats have been proposed to play a key role in directing protein-protein interactions between other proteins. These sequences in yTAFI90 could be important in mediating specific protein-protein interactions with other TAF11s, RNA polymerase II, basal factors, and/or other transcriptional regulatory proteins, either activators or repressors of transcription, as exemplified by the Tuplp/Ssn6p-a2 system (42). TAF90 was also cloned, sequenced, and noted to resemble dTAF1180 by Reese etal. (29). yTAFI60, the homolog of dTAF,,60 and hTAFI70 (9), has an overall acidic charge and may contain a leucine-zipper motif in its leucine-rich Cterminal region (Fig. 2C). The N terminus of yTAF1160 is 47% similar to both dTAFII60 and hTAF1170. Yeast TAFu-Encoding Genes Are Essential for Yeast Cell Viability. To test whether the TAF genes were essential for yeast cell viability, large regions within plasmid clones carrying each yTAF-encoding gene (TAF130, TAF90, or TAF60) were deleted and replaced with TRPJ sequences (43). The disrupting fragments were then purified and individually introduced into diploid yeast strains (44). The resulting Trp prototrophs were sporulated, and tetrads were dissected. In all three cases,
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Biochemistry: Poon et aL
Proc. Natl. Acad. Sci. USA 92 (1995)
viability segregated 2+:2- with no Trp+ spores being recovered, indicating that each cloned TAF gene is essential for viability (data not shown). To obtain viable haploid yeast strains containing the individual disrupted TAF genes, a URA3-based plasmid containing the appropriate TAF gene was introduced into the corresponding diploid disrupted strain, followed by sporulation and tetrad dissection. Those tetrads which gave rise to four viable spores were isolated and analyzed for phenotype. In each case, germinated spores that were Trp+ were also Ural (data not shown). Further, cells which were Ura+Trp+ were also 5-f luoroorotic acid sensitive (45). TSM1, which encodes yTAF11150, is known from the work of others to be an essential yeast gene (39). Together, these genetic experiments clearly indicate that TAF function is crucial for cell viability. Yeast TAF-Encoding Genes Encode Bona Fide TAFs. To unequivocably show that the four genes we have identified actually encode yTAFs, we performed a series of coimmunoprecipitation experiments to show that our TAF11s consistently copurify with yTBP and vice versa. To accomplish this, we engineered the DNA sequences encoding three copies of the influenza virus HA epitope (aa sequence YPYDVPDYA) into the TAF150, TAF130, TAF90, and TAF60 genes, as well as into the gene encoding yTBP. The epitope-tagged yTAFIi130-, yTAFIi90-, yTAF1160-encoding genes, as well as the epitopetagged yTBP-encoding gene, each resident on a HIS3, CENI ARS plasmid, were then separately exchanged for the corresponding URA3-marked, plasmid-borne, wild-type yTAF- or yTBP-encoding genes by using the plasmid shuffle technique (45) in strains carrying null chromosomal mutations of the cognate genes. The chromosomal copy of the gene encoding
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tag (as in ref. 22). The five resulting strains thus contained only the epitope-tagged version of these genes. Protein fractions prepared from these yeast strains were used in immunoprecipitation experiments (22), along with a wild-type yeast control strain which did not contain any HA-tagged genes. Using affinity-purified polyclonal rabbit anti-yTBP antibodies (Abs) for immunoprecipitations and the 12CA5 monoclonal Ab (mAb) (which recognizes the HA epitope) as the antibody for immunodetection, we found that HA-yTAF,1150, HAyTAF,1130, HA-yTAF1190, and HA-yTAF60 each coimmunoprecipitated with yTBP (Fig. 3A). Control immunoprecipitation reactions showed that, as expected, no 12CA5-reactive proteins were immunoprecipitated from the nontagged yeast cell extract, while HA-TBP was immunoprecipitated but only from the strain expressing the HA-tagged TBP (Fig. 3A, left two lanes). In the converse experiment, by using 12CA5 mAb for immunoprecipitation, followed by anti-yTBP Ab for immunodetection, yTBP coimmunoprecipitated with all of the HA-tagged TAF11s: HA-yTAFI150, HA-yTAF,1130, HA-
Z
,
Yeast expressing:
yTAF11150 was also engineered to express this same epitope
-i"
