Identification of a Yeast Transcription Factor IID Subunit,TSG2/TAF48*

Received for publication, February 29, 2000, and in revised form, March 30, 2000Published, JBC Papers in Press, April 4, 2000, DOI 10.1074/jbc.M001635200

Joseph C. Reese‡, Zhengjian Zhang, and Harsha Kurpad

From the Department of Biochemistry and Molecular Biology, The Pennsylvania State University,University Park, Pennsylvania 16802

The RNA polymerase II general transcription factorTFIID is a complex containing the TATA-binding pro-tein (TBP) and associated factors (TAFs). We have useda mutant allele of the gene encoding yeast TAFII68/61pto analyze its function in vivo. We provide biochemicaland genetic evidence that the C-terminal a-helix ofTAFII68/61p is required for its direct interaction withTBP, the stable incorporation of TBP into the TFIIDcomplex, the integrity of the TFIID complex, and thetranscription of most genes in vivo. This is the first evi-dence that a yeast TAFII other than TAFII145/130 inter-acts with TBP, and the implications of this on the inter-pretation of data obtained studying TAFII mutants invivo are discussed. We have identified a high copy sup-pressor of the TAF68/61 mutation, TSG2, that has se-quence similarity to a region of the SAGA subunit Ada1.We demonstrate that it directly interacts with TAFII68/61p in vitro, is a component of TFIID, is required for thestability of the complex in vivo, and is necessary for thetranscription of many yeast genes. On the basis of thesefunctions, we propose that Tsg2/TAFII48p is the histone2A-like dimerization partner for the histone 2B-likeTAFII68/61p in the yeast TFIID complex.

Initiation of RNA polymerase II transcription is regulatedthrough the concerted actions of general transcription factors,which bind near the polymerase start site, and specific tran-scriptional activator proteins, which bind at more distant sites(1–3). Sequence-specific gene regulatory proteins are thoughtto target multiple components of the general transcriptionalmachinery by direct protein-protein interactions or throughcoactivator proteins (3–4). Much focus has been placed onTFIID, which is composed of the TATA-binding protein (TBP)1

and a collection of tightly associated factors commonly referredto as TAFIIs (4, 5). A plethora of biochemical analyses of mam-malian and Drosophila TFIID indicates that TAFIIs play acrucial role in promoter recognition and may be a target ofsome activator proteins (4, 5).

The identification of yeast TAFIIs (6, 7) and the subsequentanalysis of mutants in vivo (8–10) altered our understanding ofthe role of TAFIIs in the regulation of transcription. Despiteforming a discrete complex, mutation of different TAFII genescan have very different effects on the structure and function ofthe TFIID complex (11). Mutation or depletion of seven differ-ent yeast TAFIIs failed to result in global changes in geneexpression (8–10, 12). Most notably, mutation or depletion ofTAFII145p, the dTAFII250 homologue, affects only a small por-tion of the genome (9, 10, 12–14). In contrast, inactivation oftemperature-sensitive mutants of yeast TAF68/61, TAF60,TAF40, TAF25, and TAF17 resulted in much broader tran-scriptional phenotypes (15–20). The interpretation of the re-sults obtained using TAFII mutants has been complicated bythe presence of certain TAFIIs in the SAGA histone acetyl-transferase complex (21, 22). It has been proposed that thebroad transcriptional phenotypes of some TAFII mutants mayresult from their presence in both TFIID and the SAGA histoneacetyltransferase complex (17). However, although presence ofsome TAFIIs in both TFIID and SAGA may contribute tobroader transcriptional phenotypes, it cannot solely explainthese effects. Mutations in TAF40, whose translation product isbelieved to be part of TFIID but not SAGA, cause a broadtranscriptional phenotype (19).

There is a strong correlation between the severity of theeffects of TAFII mutations on the integrity of the TFIID com-plex in vivo and the fraction of the genome affected by theirinactivation. All TAFIIs whose inactivation leads to decreasesin the expression of a large percentage of the genome cause aconcurrent reduction of additional TAF subunits and TBP invivo. In particular, the TAFII genes with similarities to the corehistones are important for the structure of TFIID in vivo. Incontrast, mutation or depletion of the two largest yeast TAFIIs,TSM1p or TAFII145p, has relatively minor effects on TFIIDstructure (9, 10). This is surprising considering thatyTAFII145p (dTAFII250) is the subunit that binds to TBP withhighest affinity (6, 22–24), and dTAFII250 is absolutely essen-tial to form a functional TFIID complex in biochemical recon-stitution experiments (25). A logical explanation for these ob-servations is that additional TFIID subunits bind to TBP in theabsence of TAFII145p, and that the histone-like TAFIIs aremore important for TFIID structure.

Structural and biochemical studies on metazoan TAFIIs re-vealed that three TAFIIs adopt a structure similar to the his-tone fold of the core histones and are capable of forming dimerpairs (26–28). The structure of the dTAFII60/dTAFII40(yTAFII60/yTAFII17) dimer resembles that of histone H3/H4 inthe 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 iden-tified as hTAFII135 (29). These same studies identified the

* This work was supported in part National Institutes of HealthGrant GM58672 (to J. C. R.). The costs of publication of this article weredefrayed in part by the payment of page charges. This article musttherefore be hereby marked “advertisement” in accordance with 18U.S.C. Section 1734 solely to indicate this fact.

‡ To whom correspondence should be addressed: Dept. of Biochemis-try and Molecular Biology, Pennsylvania State University, 203 Alt-house Laboratory, University Park, PA 16802-4500. Tel.: 814-865-1976;Fax: 814-863-7024; E-mail: jcr8@psu.edu.

1 The abbreviations used are: TBP, TATA-binding protein; TAFIIs,RNA polymerase II-specific TBP-associated factors; SAGA, SPT-ADA-GCNS histone acetyltransferase complex; ts2, temperature-sensitiveminus; YP, yeast extract-peptone medium; PAGE, polyacrylamide gelelectrophoresis; GST, glutathione S-transferase; HA, hemagglutinin.

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 275, No. 23, Issue of June 9, pp. 17391–17398, 2000© 2000 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.

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SAGA component Ada1, as the H2A partner of yTAFII68/61p inthe SAGA complex. Unfortunately, there is no yeast proteinthat shows extensive sequence homology to hTAFII135, leavingthe dimerization partner of TAFII68/61p in the yTFIID complexunidentified.

The analysis of yeast TAFII68/61 contained in this report hasaddressed two important issues regarding the function of thisgene and its role in the yTFIID complex. First, we have iden-tified the putative C-terminal a-helix of TAFII68/61p as impor-tant for the interaction of TBP with the yTFIID complex, indi-cating that TAFII145p is not the only subunit capable ofbinding to TBP. Second, we have isolated a gene, TSG2/MPT1,as a suppressor of a TAF68/61 mutation and demonstrate thatit can stabilize the mutant TFIID complex in vivo. Finally, wehave identified Tsg2/TAFII48p as a bona fide TFIID subunitand provide genetic, molecular, and biochemical evidence thatit is the dimerization partner for TAFII68/61p in the TFIIDcomplex.

MATERIALS AND METHODS

Isolation of Temperature-sensitive Mutants and High Copy Suppres-sors—pRS414-TAF68/61 was mutagenized in vitro for 60–120 min at70 °C using hydroxylamine, purified and transformed directly intoYJR20 (Mat a; Dtaf68:hisg; ade2–11; his3–11, 15; leu2–3, 112; ura3–1;trp1–1; can1–100; [RS416-TAF68/61]). Tryptophan prototrophs werereplica plated onto 5-fluoroorotic acid-containing media at 23 °C and37 °C, and mutants that supported growth at 23 °C, but not 37 °C, wereisolated for further analysis. The ts2 phenotypes were verified to beplasmid-linked by recovery of the plasmids, re-transformation intoYJR20, and rescreening at the indicated temperatures. Mutants thatceased growth at #2 doubling times were analyzed further. The strainYJR21–9 (taf68–9) was transformed with a Yep13-based high copygenomic library obtained from the ATTC. Approximately 40,000 trans-formants were plated at 23 °C for 16 h and than transferred to 37 °C for2–5 days. Colonies were isolated, and the plasmids were recovered fromthe cells and reintroduced into YJR21–9. Plasmids that conferredgrowth at 37 °C were sequenced, and the open reading frame conferringthe suppression phenotype was identified by subcloning into pRS426and rescreening in YJR21–9.

Conditional Depletion of TSG2—The plasmid pGAL1-TSG2/TAF48 wasconstructed by cloning the polymerase chain reaction-amplified coding se-quence of TSG2/TAF48 into the vector pSW107 (10) and integrated at theTRP3 locus of the strain BY4705 (Mat a; ade2D::hisg; his3D200; leu2D0;lys2D0; met15D0; trp1D63; ura3D0). After verification, the chromosomalTSG2/TAF48 gene was deleted by homologous recombination using a po-lymerase chain reaction-based strategy resulting in the strain YJR475 (Mata; ade2D::hisg; his3D200; leu2D0; lys2D0; met15D0; trp1D63; ura3D0;Dtsg2::LEU2; trp3::GAL1p-HA-TSG2::HIS3). Cultures of YJR475 weregrown to an optical density of 1.0 in YP plus galactose, collected by centrif-ugation, washed once in YP, and grown in YP plus dextrose for the timesindicated in the Fig. legends.

Analysis of RNA and Protein Levels and Immunoprecipitation—Cellswere grown to midlog phase in YP plus dextrose (unless indicatedotherwise) and shifted to a 37 °C shaking water bath. Aliquots weretaken from the culture prior to temperature shift and at 0.5, 1, 2, and4 h thereafter. After centrifugation, cells were washed in ice-cold STE(Tris-EDTA plus 100 mM NaCl) and separated into two parts for theisolation of RNA and protein extract preparation. RNA was prepared (8,10) and analyzed by S1 nuclease protection (30) 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 mM sodium orthovanadate, 25 mM

sodium fluoride, and protease inhibitors (6). We find that the additionof NaCl to .0.6 M significantly aids in the extraction of TAFs and othertranscription factors. 20 mg of whole cell extract protein was fraction-ated on SDS-PAGE gels and transferred to nitrocellulose in bufferscontaining and lacking SDS, depending on the TAFs to be analyzed.Antibodies and Western blotting were described (10).

Mid-scale extracts were prepared from 1-liter cultures of cells grownat the permissive temperature as follows. Cells were collected by cen-trifugation and washed in ice-cold STE plus 1 mM benzamidine HCl and0.2 mM phenylmethylsulfonyl fluoride and frozen. One cell pellet vol-ume of buffer T containing 0.3 M potassium acetate plus 0.1% NonidetP-40 and protease inhibitors (6) and 1 volume of glass beads wereadded, and the cells were broken by vortex mixing for 6 cycles of mixing

and cooling. The beads and debris were separated by low speed centrif-ugation, and the supernatant was further clarified by centrifugationtwice for 30 min at 30,000 3 g. TFIID was immunoprecipitated from 1ml of extracts diluted to 2–3 mg/ml protein with 0.3 M buffer T asfollows. Antiserum was added for 2–4 h on ice, followed by an overnightincubation with protein A-agarose beads (Repligen). Protein A beadswere collected by low speed centrifugation and washed three times for10 min each in buffer T containing the concentrations of potassiumacetate indicated in the figure legends. After a brief wash in buffer Tcontaining 0.1 M potassium acetate, proteins were eluted in SDS-PAGEloading buffer.

TBP and Tsg2 Binding Studies—The region of TAF68 and taf68–9encoding the conserved C-terminal portion of the protein was amplifiedby polymerase chain reaction and cloned into the BamHI and EcoRIsites of pGEX-1. Glutathione S-transferase (GST) proteins were pro-duced in XA90 cells and purified on glutathione-agarose beads, andBradford assays and Coomassie Blue-stained SDS-PAGE gels moni-tored protein concentrations. Equal amounts of GST fusion protein wasbound to the beads. Yeast TBP and Tsg2/TAF48p was in vitro trans-lated from pet15-yTBP (31) and bluescript-TSG2/TAF48, respectively,using a wheat germ transcription and translation (TnT) kit from Pro-mega. Three micrograms of GST fusion protein bound to 5 ml of gluta-thione beads were incubated with 20 ml of wheat germ extract and 80 mlof 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% NonidetP-40) at 4 °C for 60 min with agitation. Afterward, 200 ml of ice-coldbinding buffer was added, and the beads were collected by low speedcentrifugation. The beads were washed 4 times for 5 min each with 300ml of binding buffer and finally eluted with SDS-PAGE loading buffer.The bound proteins were separated by SDS-PAGE, stained with Coo-massie Blue, treated with En3Hance (NEN Life Science Products),dried, and exposed to film.

RESULTS

Isolation and Characterization of TAF68/61 Temperature-sensitive Mutants—In vitro random chemical mutagenesis andplasmid shuffling was used to isolate three TAF68/61 mutantsthat could not support growth at 37 °C (Fig. 1A). The sequenceof the mutants was determined, and it was found that all three

FIG. 1. Isolation and characterization of TAF68/61 tempera-ture-sensitive mutants. A, strains containing wild type (YJR21–0) orthree temperature-sensitive alleles of TAF68/61 (YJR21–9, YJR21–22,and YJR21–31) were streaked onto YP-dextrose plates and incubatedfor 3 days at 23 °C or 37 °C (above). Western blot analysis of the mutantstrains reveals that the TAF68/61 mutants are truncations (below). B,the temperature-sensitive mutants were sequenced, and the position ofthe mutant verifies the truncation. The position of the truncation inrelation to the conserved a-helices comprising the histone H2B fold isindicated above. The black box indicates the region that is conservedamong all identified homologues and resembles histone H2B. Thehatched box indicates the region shown to interact with Ada1.

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alleles contained a single nucleotide change at nucleotide 1456(ATG 5 11) resulting in the change from a tryptophan residueto a stop codon at amino acid 486. The expressed protein istruncated for the last 54 amino acids (Fig. 1B). It is interestingto note that the only tight ts2 mutants we isolated out of a total20,000 colonies screened contained the same mutation. More-over, two ts2 alleles of TAF68/61 described in another reportalso contained truncations at and near this region (16). Trun-cation at residue 486 removed the C-terminal a-helix (Fig. 1B)but retained the three a-helices comprising the histone fold(32).

Inactivation of three different yeast TAFII mutants results ina specific cell cycle arrest phenotype (8, 10); we therefore ana-lyzed the cell cycle phenotypes of the taf68–9 mutant followinggrowth at the restrictive temperature. We found that shiftingthe taf68–9 mutant to the restrictive temperature caused arapid cessation of growth with no specific cell cycle phenotypes(not shown).

Inactivation of the taf68–9 Allele Causes the Rapid Degrada-tion of TFIID Subunits and a Broad Transcription Pheno-type—A previous study showed that inactivation of tempera-ture-sensitive mutants of TAF68/61 resulted in the reducedtranscription of a large number of yeast genes (16). We there-fore examined the effects of taf68–9 inactivation on the tran-scription of yeast genes to verify the phenotype in our strain. Awild type strain, the taf68–9 mutant, and as a control, a tem-perature-sensitive RNA polymerase II mutant (rpb1–1), weretransferred to the restrictive temperature, and RNA was iso-lated at various times. The mRNA levels of seven representa-tive genes were analyzed by S1 nuclease protection. Within 1 hof temperature shift, we found significant reductions in themRNA levels of a number of RNA polymerase II-transcribedgenes (Fig. 2A and data not shown), but only minor effects wereobserved on the levels of SPT15 and DED1 mRNA. The expres-sion of an RNA polymerase III-transcribed gene (tRNA) wasonly mildly affected at later time points (Fig. 2A). We nextexamined the effect of the inactivation of taf68–9 on the ex-pression of a number of highly induced genes. The wild typeand taf68–9 mutant was preincubated at 37 °C for 1 h and thentreated under the inducing conditions described in the figurelegends. Growth of the taf68–9 mutant at 37 °C prevented theinduction of the RNR2 gene by DNA damage and the derepres-sion of the SUC2 gene (Fig. 2B). Moreover, we found that theuninduced levels of SUC2 and RNR2 were reduced in themutant. However, inactivation of taf68–9 did not affect theinduction of all genes tested. Induction of the SSA4, CUP1, andCTT1 were not significantly reduced by the inactivation of thetaf68–9 mutant but were strongly affected by mutations in thelarge subunit of RNA polymerase II (Fig. 2B).

Incubation of certain TAFII mutants at the restrictive tem-perature results in the disruption of the TFIID complex and thedegradation of TAFII subunits in vivo (10, 16, 19, 20); therefore,we analyzed the effects of shifting the taf68–9 mutant to therestrictive temperature on the steady state levels of TAFII

protein. We found that incubation of the mutant at 37 °Ccaused a rapid reduction in the levels of TBP, TAFII145p, andTAFII47p and, to a lesser degree, TAFIIs 90p, 68p, 60p, 25p,and 17p (Fig. 2C and data not shown). In contrast, Sua7p, anuclear protein not associated with TFIID or SAGA, was notaffected. It appears that the TAFIIs not shared between TFIIDand the SAGA complex (21) are less stable in the mutant.Shifting a TBP (tbp1–1; Fig. 2C) or a RNA polymerase II ts2mutant (not shown) to the restrictive temperature failed tocause similar reductions in TAFII protein levels, indicating thatthe reductions in TBP and TAFII protein in the taf68–9 mutantis largely at the level of protein turnover. Interestingly, growth

of the TBP mutant at 37 °C resulted in the rapid turnover ofTBP but not other subunits in the TFIID complex, indicatingthat TBP is not required for the stability of TAFIIs.

TAF68/61p Binds TBP in Vitro—Our results above demon-strate that inactivation of temperature-sensitive mutants ofTAF68/61 causes a loss of TBP protein within the cell that isfaster and more severe than what is observed in TAF145 mu-tants (10). This result is surprising in light of the fact thatTAFII145p was identified as the single subunit of TFIID thatbinds to TBP with high affinity by Far Western blotting (6). Alogical explanation for these findings is that in the absence ofTAFII145p another TFIID subunit binds to TBP and protects itfrom degradation. The human homologue of TAFII68/61p(hTAFII20) can bind to TBP in vitro (31), and it is likely thatTAFII68/61p has this function. To test this hypothesis, weexpressed the conserved C-terminal portion (amino acids 330–539) of TAFII68/61p as a GST derivative and used it in pulldown assays to identify an interaction with in vitro translatedTBP. This region alone retains all the essential functions of thefull-length TAFII68/61protein because it can fully complementa TAF68/61 gene disruption (33). A GST derivative containingthe activation domain of the herpesvirus protein VP16 wasused as a positive control in our experiments. TBP was capableof interacting with the C terminus of TAFII68/61p; moreover, itbound to TBP more strongly than GST-VP16 (Fig. 3B). How-ever, when the assay was carried out using a GST derivativecorresponding to the taf68–9 mutation, GST-taf68–9C (aminoacids 330–485), we found the mutant was defective for itsability to interact with TBP in vitro (Fig. 3B).

The binding of TBP to the N terminus of TAFII145p anddTAFII230 has been extensively characterized (6, 22, 23, 34).To address the significance of the TBP-TAFII68/61 interaction,we compared the TBP binding ability of GST-TAFII68C versusGST-TAFII145N (amino acids 5–230) under identical condi-tions. The results contained in Fig. 3C clearly show that TBPinteracted equally well with the C terminus of TAFII68/61p andthe N terminus of TAFII145p. Our TBP binding studies dem-onstrate that the C-terminal a-helix (485–539) of TAFII68/61pis required for its ability to bind to TBP and that this interac-tion is comparable with that of the well characterizedTAFII145p-TBP interaction.

We next sought to determine if the C terminus of TAFII68/61p is important for the interaction of TBP with the TFIIDcomplex by comparing the ability of TFIID to co-immunopre-cipitate with TBP in extracts of wild type and mutant cells. Forthis experiment, extracts were prepared from cells grown at thepermissive temperature because incubation at 37 °C wouldresult in the depletion of TAFs and ambiguous results. Wefailed to detect any differences in the co-immunoprecipitationof TFIID in extracts from wild type and mutant cells under ourstandard medium salt conditions (Fig. 3D and data not shown).Since TAFII145p is present in these complexes, it is likely thatits interaction with TBP is obscuring the detection of anydifferences between the wild type and mutant TFIID complexesthat would otherwise be apparent in vivo. Therefore, we per-formed our analysis under conditions that disrupt the TBP-TAFII145Np interaction and accentuate the contributions ofother TBP-TAF interactions. The TAFII145Np-TBP interactionis sensitive to potassium acetate concentrations above 0.3 M

(23), and yet the binding of TBP to the intact TFIID complex isresistant to salt concentrations above 1 M potassium acetate (6,7), indicating that TBP makes contacts with additional TAFsubunits. Based on our results above, a likely candidate isTAFII68/61p. TFIID was immunoprecipitated using anti-TBPantibodies in extracts adjusted to potassium acetate concentra-tions of 0.3 to 1.0 M, and the immune complexes were washed

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with buffers of the corresponding ionic strengths. TFIID sub-units co-immunoprecipitated with TBP in extracts from wildtype cells in buffers containing all concentrations of potassiumacetate (Fig. 3D). A noticeable reduction was observed at saltconcentrations above 0.6 M in the wild type extracts, likelyresulting from the disruption of the TBP-TAFII145Np interac-tion. However, the recovery of TFIID subunits was stronglyaffected in the mutant extracts when salt concentrations wereabove 0.6 M potassium acetate (Fig. 3D). The increased potas-sium acetate concentrations did not affect the ability of theantibody to recognize TBP, because equal amounts of TBP wereprecipitated under all salt concentrations. The data presentedin Figs. 3 and 4 support a role for the C-terminal a-helix ofTAFII68/61p in the binding of TBP and stabilizing its interac-tion with the TFIID complex.

Isolation of TSG2/MPT1 as a High Copy Suppressor of thetaf68–9 Mutant—In an attempt to identify the functions ofTAF68/61, we screened for high copy number suppressors ofthe temperature-sensitive growth defect of the taf68–9 strain,TAF suppressor genes, TSGs.2 The gene (TSG2) conferring thestrongest suppression phenotype was identified as MPT1, anessential gene reported to have an unspecified role in proteinsynthesis.3 Expression of TSG2 from a high copy number plas-mid did not result in any obvious growth alterations in cellscontaining a wild type copy of TAF68/61; however, it didstrongly suppress the temperature-sensitive growth defects ofthe taf68–9 allele (Fig. 4). Overexpression of TSG2 did notsuppress mutant alleles of TAF145, TAF17, TAF90, TSM1, orSPT15 (TBP), indicating that the suppression is somewhatspecific for TAF68/61 (data not shown).

Inactivation of the taf68–9 allele caused the rapid turnoverof TFIID subunits and reduced mRNA levels of a number ofyeast genes (Fig. 2). We therefore examined if overexpression ofTSG2 can suppress these defects. Strains containing thetaf68–9 allele transformed with Yep13, Yep13-TAF68/61, orYep13-TSG2 were grown at the permissive temperature,

2 J. C. Reese and M. R. Green, manuscript in preparation.3 L. A. Estey and M. G. Douglas, GenBanky accession number

X79240.

FIG. 2. Effects of temperature shift on the transcription ofyeast genes and stability of the TFIID complex. A, analysis of thesteady state mRNA levels. Cultures of wild type (YJR21–0), taf68–9(YJR21–9) and an RNA polymerase II mutant (rpb1–1) were shifted to37 °C, and aliquots of cells were drawn at the times indicated above andused to prepare RNA. RNA was detected by S1 nuclease protection. B,analysis of gene induction. Cells were grown at 23 °C to log phase,shifted to 37 °C for 1 h, and then treated as follows: a 20-min heat shockat 39 °C (SSA4), 0.5 mM copper sulfate for 30 min at 37 °C (CUP1), 1 M

NaCl for 2 h at 37 °C (CTT1), 0.03% methylmethane sulfonate for 2 h at37 °C (RNR2); transfer to 0.05% dextrose for 2 h at 37 °C (SUC2). SSA4and CUP1 mRNA were detected by primer extension and S1 nucleaseprotection, respectively. CTT1, SUC2, and RNR2 mRNA were detectedby Northern blotting. scR1, an RNA polymerase III-transcribed gene,was used as a loading control. C, immunoblotting was carried out usingthe antibodies indicated on the right. Sua7 (TFIIB) was used as acontrol.

FIG. 3. The C terminus of TAFII68/61p binds TBP. A, GST deriv-atives used in this study. B, yeast TBP was synthesized by in vitrotranslation in the presence of [35S]methionine and incubated with equalamounts of GST, GST-TAF68C, GST-taf68–9C, or GST-VP16. Afterextensive washing, TBP was eluted in loading buffer, separated bySDS-PAGE, and detected by fluorography. C, comparison of TBP bind-ing by GST-TAF68C versus GST-TAF145N. D, binding of TBP to wildtype and mutant TFIID complexes. Extracts from wild type and taf68–9mutant cells were adjusted to 0.3, 0.6, 0.8, and 1.0 M potassium acetateand were incubated with preimmune rabbit serum (PI) or anti-TBPpolyclonal antibodies (aTBP). Immune complexes were recovered usingprotein A-agarose beads and were extensively washed in buffers of thecorresponding molarities of potassium acetate. Blotting for TBP con-trolled for recovery of the immune complexes.

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shifted to the restrictive temperature, and treated as describedin Fig. 2 and in the figure legends. The steady state levels ofmRNA of three yeast genes, TCM1, ACT1, and PMA1, that arestrongly affected by the inactivation of the taf68–9 mutantwere examined. Overexpression of TSG2 was capable of sup-pressing the transcriptional defects of the taf68–9 mutant, asevidenced by the increased levels of TCM1, ACT1, and PMA1mRNA (Fig. 5A). Likewise, overexpression of TSG2 partiallysuppressed the reduced expression of the RNR2 and SUC2genes (Fig. 5B). Since the inactivation of taf68–9 causes theinstability of the TFIID complex in vivo and the subsequentturnover of individual TAFs, we examined the ability of TSG2to stabilize the TFIID complex in vivo. The steady state levelsof the TFIID subunits that were least stable in the mutant,namely, TAFII145p, TAFII60p, TAFII47p, and TBP were exam-ined following temperature shift. The results in Fig. 5C showthat overexpression of TSG2 resulted in the partial stabiliza-tion of the TFIID complex in vivo, measured by the increasedstability of the individual TAFIIs following temperature shift.These results indicate that overexpression of TSG2 suppressesthe taf68–9 defect by stabilizing the TFIID complex and sup-pressing the transcriptional defects.

Identification of Tsg2 as a Subunit of the TFIID Complex,TAFII48p—Interestingly, the region between amino acids 189to 243 of Tsg2 displays significant homology to a domain ofAda1 shown to interact with TAFII68/61p (29). This region issimilar to the histone fold motif of histone 2A and is likely to bean interaction site for the histone 2B-like TAFII68/61p. Wetherefore examined the ability of Tsg2p to directly interact withTAFII68/61p in vitro. Full-length in vitro translated Tsg2 in-teracted with both GST-TAF68C and GST-taf68–9C equallywell (Fig. 6A). These results are not surprising given that thetaf68–9 mutation truncates TAFII68/61p at amino acid 485,preserving most of the Ada1 interaction domain located be-tween amino acids 414 and 490 (29). To verify that the putativehistone 2A-like domain of Tsg2 is required for its interactionwith TAFII68/61p, we in vitro translated a derivative that istruncated in this domain (1–228) and analyzed its ability to

bind to GST-TAF68C. Our results indicate that disruption ofthis region abolishes Tsg2 binding to TAFII68/61p (Fig. 6A).Moreover, we find that a major translation termination productthat retains this region binds to GST-TAF68C, but minor prod-ucts that are truncated into this region do not (Fig. 6A).

The binding of Tsg2 to TAFII68/61p in vitro and its ability tostabilize mutant TFIID complexes in vivo suggests that it is thedimerization partner of TAFII68/61p in the TFIID complex. Toverify this, we constructed a strain expressing an epitope-tagged version of TSG2 under control of the GAL1 promoterand immunoprecipitated TFIID from extracts prepared fromthese cells using antibodies to known TFIID subunits. Epitope-tagged Tsg2 (HA-Tsg2) and other TFIID subunits were co-immunoprecipitated by antibodies recognizing TBP,TAFII145p, and TAFII90p (Fig. 6B). Most notably, HA-Tsg2pwas precipitated using antibodies to the TFIID-specific TAFII,TAFII145p (6, 7, 21). Attempts at performing the reciprocalexperiment of using antibody recognizing the tagged version of

FIG. 4. Overexpression of TSG2 suppresses the temperature-sensitive growth phenotype of the taf68–9 mutant. Wild type(TAF68/61) and mutant (taf68–9) strains were transformed withYep13, Yep13-TAF68, or Yep13-TSG2 and streaked onto yeast syn-thetic drop-out (SD)-leu media and incubated for 2 days at 37 °C or 3days at 23 °C.

FIG. 5. Overexpression of TSG2 stabilizes mutant TFIID com-plexes in vivo and suppresses the transcriptional defects. A,YJR21–9 was transformed with the plasmids indicated within the fig-ure and grown in yeast synthetic drop-out medium at the nonpermis-sive temperature to the middle of logarithmic growth (0), then shifted to37 °C, and aliquots of cells were removed for RNA isolation and proteinextraction at 1-h intervals. RNA was detected by S1 nuclease protec-tion. B, wild type (TAF68/61, YJR21–0) or mutant (taf68–9, YJR21–9)cells were transformed with either Yep13 or Yep13-TSG2 and grown toan optical density of 0.4 at 23 °C, transferred to 37 C for 1 h, and thentreated with 0.03% methylmethane sulfonate (MMS; 1, above) or 2%dextrose (R, below) transferred to prewarmed media containing 0.05%dextrose (D, below) for an additional 2 h. C, whole cell extracts wereprepared from an aliquot of cells from panel A and used to measure TAFprotein levels by Western blotting.

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HA-Tsg2 to co-immunoprecipitate TFIID subunits have notbeen successful because the N terminus of Tsg2 is inaccessiblein its native state (data not shown). The sequence similaritybetween Tsg2 and Ada1 suggests that they interact withTAFII68/61p in TFIID and SAGA, respectively. However, todirectly verify that Tsg2 is not part of the SAGA complex, weperformed immunoprecipitations using antibodies to Spt7p, acomponent of the SAGA complex (21). The result in Fig. 6Cshows that antibodies to Spt7 can immunoprecipitateTAFII90p, TAFII68p, and TAFII25p, three TAFIIs found inSAGA, but not TAFII145p or HA-Tsg2. These data indicate thatTsg2p interacts with TAFII68/61p and is a bona fide subunit of

the TFIID complex. From here on, TSG2 will be referred to asTSG2/TAF48.

TSG2/TAF48 Is Broadly Required for Transcription in Yeastand for the Stability of the TFIID Complex in Vivo—The se-quence similarity between Tsg2 and histone 2A and its abilityto interact with the histone 2B-like TAFII68/61p, indicates thatit belongs to a class of TAFII genes known as the “histone TAFs”(32, 35). Inactivation of temperature-sensitive mutants ofTAF68/61 and other histone TAFIIs results in the destabiliza-tion of TFIID and a decrease in the transcription of many yeastgenes in vivo (see above and Ref. 16); therefore, we expectedthat if Tsg2/TAFII48p is the dimerization partner of TAFII68/61p it would be required for these two functions as well. To testthis hypothesis, a strain containing the sole copy of TSG2/TAF48 under control of the dextrose-repressible GAL1 pro-moter was constructed and used to analyze the effect of Tsg2/TAFII48p depletion on the transcription of yeast genes and thestability of the TFIID complex in vivo. This strain cannot growon dextrose-containing plates and stops proliferating in liquidculture after transfer from galactose- to dextrose-containingmedium (Fig. 7A). Cells depleted of Tsg2/TAFII48p ceased pro-liferation at every stage of the cell cycle, indicating that it is notspecifically required for cell cycle progression (data not shown).

To verify the depletion of Tsg2/TAFII48p, immunoblot anal-ysis was performed in the conditional expression strain and itscogenic wild type. Fig. 7B shows that depletion of Tsg2/TAFII48p occurred within 8 h after being transferred to dex-trose-containing medium. In addition, we found the concurrentloss of the TFIID subunits Tsm1p, TAFII145p, TAFII68p,TAFII47p, and TBP (Fig. 7B) and, to a lesser degree, TAFII90p,TAFII60p, TAFII25p, and TAFII17p. In contrast, proteins notpart of TFIID, such as Sua7p, were unchanged. Similar to whatwe observed in strains depleted of TAFII68/61p (10) and in thetaf68–9 strains (Fig. 2), the turnover of TAFIIs found in TFIID,but not SAGA, underwent a more rapid and drastic reductionin steady state protein levels. These data demonstrate that,like TAF68/61 and other histone TAFIIs, TSG2/TAF48 is re-quired for the stability of the TFIID complex in vivo.

The loss of function of TAFII genes can result in either broaddefects in transcription or the reduced expression of a limitednumber of genes (11). To determine which of these phenotypesresult from Tsg2/TAFII48p depletion, we analyzed the transcrip-tion of a variety of yeast genes before and at 4-h intervals afterthe transfer of cells to dextrose-containing medium. Fig. 8Ashows that the levels of DED1, TCM1, ENO2, ACT1, ADH1,CLN2, CLB2, CLN3, and RPS5 mRNA were significantly re-duced. The reduction in transcription strongly correlated withthe depletion of Tsg2/TAFII48p and other TAFIIs, with the notedexception of ADH1. Not all genes examined were affected byTsg2/TAFII48p depletion; the RNA polymerase II-transcribedgenes MET19 and TRX2 and the RNA polymerase III-tran-scribed genes tRNAmet and scR1 remained unchanged. We nextexamined the effect of Tsg2/TAFII48p depletion on the inductionof genes by first growing the cells for 12 h in dextrose to allow forthe depletion of the TAFII and then treating the cells with con-ditions that activate the genes of interest (see figure legends).The induction of heat shock-inducible SSA4, copper-inducibleCUP1, diamide-inducibleTRX2, and stress-inducible CTT1 wasunaffected (Fig. 8B). However, depletion of Tsg2/TAFII48p abro-gated the induction of the DNA damage-inducible RNR2 andRNR3 and the cycloheximide-inducible PDR5 (Fig. 8B). More-over, we observed a reduction in uninduced levels of RNR2.These data indicate that TSG2/TAF48 is globally, but not gen-erally, required for the expression of yeast genes, and depletion ofTsg2/TAFII48p has similar phenotypes as those resulting fromthe inactivation of the taf68–9 allele.

FIG. 6. Tsg2 interacts with TAFII68/61p and the TFIID com-plex. A, interaction between in vitro translated Tsg2 and GST-TAF68C.In vitro translated full-length Tsg2 (1–388) or a mutant truncated in thehistone 2A-like domain (1–228) was incubated with GST, GST-TAF68C,or GST-taf68–9C, and Tsg2 binding was detected as described under“Materials and Methods.” The location of the region homologous toAda1 and resembling histone 2A is indicated below in the hatched box.The asterisk indicates the position of a major translation terminationproduct. B, Tsg2 co-immunoprecipitates with TFIID subunits. Pre-immune serum (PI) or antibodies to TBP (aTBP), TAFII145p(aTAF145), or TAFII90p (aTAF90) were used to co-immunoprecipitateHA-Tsg2 from whole cell extracts prepared from strain YJR375. Thepresence of TAFs and Tsg2 in the immunoprecipitates was detected byWestern blotting. C, Tsg2 is not associated with the SAGA complex.Antibodies to Spt7 were used in co-immunoprecipitation the SAGAcomplex from whole cell extracts (WCE).

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DISCUSSION

TAFII68/61p and its higher eukaryotic orthologs have strongsequence similarity to the conserved region of histone H2B (4,32, 35). The primary amino acid sequence of the histone-likeTAFII68/61p, TAFII60p, and TAFII17p predicts that they con-tain a histone-fold motif and may form a higher order structuresimilar to the histone octamer (4, 26–28, 32). The originalmodel for an octamer-like structure in TFIID depictedhTAFII20/dTAFII30a/yTAFII68/61 as forming a homodimer tofulfil the requirements for octamer formation, due to the lack ofa known dimerization partner (32). The identification of Ada1

and Tsg2/TAFII48p as the dimerization partners of TAFII68/61p in SAGA (29) and TFIID, respectively, makes this scenariounlikely. Tsg2/TAFII48p was not detected in previous prepara-tions of purified TFIID because of a co-migrating contaminant(6, 7); however, Tsg2/TAFII48p was recently identified as asubunit TFIID in preparations of highly purified TFIID (39).The primary amino acid sequence of a region of Tsg2/TAFII48pis highly homologous to a domain of Ada1 shown to het-erodimerize with the histone fold of TAFII68/61p (29). We haveverified that this region containing the putative histone fold isrequired for its direct interaction with TAFII68/61p. The ge-

FIG. 7. Conditional expression of TSG2/TAF48. A, a strain containing the sole copy of TSG2 under control of the GAL1 promoter (YJR375;GAL1-TSG2) and its co-genic wild type (BY4705; TSG2) were spread onto YP 1 2% dextrose (upper right) or YP 1 2% galactose (upper left). Plateswere incubated at 30 °C for 3 days. Cells were grown in liquid YP 1 2% galactose to mid-log phase growth at 30 °C, harvested, washed in YP, andseeded into YP 1 2% dextrose at an optical density of 0.1 (below, right). Cells were maintained at an optical density (OD) of 0.3–0.8 during theexperiment by diluting cultures with YP 1 2% dextrose wherever appropriate. The optical densities of cultures of BY4705 (closed circles) andYJR375 (open squares) were plotted on a logarithmic scale versus time. Cell growth was monitored every 2 h, and aliquots of cells were drawn every4 h for protein extraction and RNA isolation (see below). B, Western blotting for TFIID subunits. Note: the growth curve presented was from thecells used to prepare protein extracts in panel B and RNA for Fig. 8A.

FIG. 8. Effects of Tsg2/TAFII48p depletion on RNA polymerase II transcription. A, RNA was isolated from strains BY4705 (TSG2/TAF48) and YJR475 (GAL1-TSG2/TAF48) that were grown in YP 1 2% galactose (0) and then at 4-h intervals after being transferred to YP 12% dextrose (4, 8, 12, 16). RNA levels were detected by S1 nuclease protection (DED1, TCM1, ENO2, ACT1, ADH1, MET19, tRNAmet) or Northernblotting (TRX2, CLN2, CLB2, CLN3, RPS5, and scR1). B, cells were grown in YP 1 2% galactose, then an aliquot was then removed (G), and thecells were transferred to YP 1 2% galactose for 12 h (D). Afterward, the culture was divided into pairs, one was left untreated (D, 2), and the otherwas treated under the appropriate induction conditions in (D, 1). For the induction of the genes indicated, cells were treated as follows: SSA4, 20min at 39 °C; CUP1, 30 min with 0.5 mM copper sulfate; TRX2, 1 h with 1.5 mM diamide; CTT1, 2 h with 1M NaCl; RNR2 and RNR3, 2 h with0.03% methylmethane sulfonate (MMS); and PDR5, 1 h with 0.2 mg/ml cycloheximide (CHX).

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netic evidence presented here showing that overexpression ofTSG2/TAF48 suppresses mutations in TAF68/61 supports aninteraction between TAFII68/61p and Tsg2/TAFII48p in vivo.The growth defects of temperature-sensitive mutants of thehistone-like TAF17, TAF40, and TAF60 genes are suppressedby the overexpression of their respective dimerization partners(16, 19). We have not only shown that TSG2/TAF48 canstrongly suppress the growth defects of the taf68–9 mutant buthave also demonstrated that it stabilizes the mutant TFIIDcomplex in vivo. TSG2/TAF48 is broadly, but not universally,required for the transcription by RNA polymerase II and isnecessary for the stability of the TFIID complex in vivo. Thesephenotypes are strikingly similar to those of temperature-sen-sitive mutants of TAF68/61 (Ref. 16 and this study). Based onour biochemical, genetic, and molecular evidence, we proposethat Tsg2/TAFII48p is the dimerization partner of TAFII68/61pin the TFIID complex. Since Tsg2/TAFII48p is a component ofTFIID, but not SAGA, its broad transcriptional phenotype can-not be explained by redundancy between these two complexes.These results complement and strengthen the observations ofKomarnitsky et al. (19) that some TFIID-specific TAFIIs arerequired for the transcription of most genes.

One of the most highly debated issues in gene regulation isthe contribution of individual TAFs in the regulation of geneexpression in vivo and what accounts for the phenotypes oftheir mutants. Earlier models of metazoan TFIID structurewould have predicted that yTAFII145 (dTAFII250) and Tsm1(dTAFII150) would comprise the “scaffold” for the entire yeastcomplex (5, 25) and, thus, would be the most important sub-units for the structure and function of the TFIID complex.Besides its large size, TAFII145 was the only yeast TFIIDsubunit known to bind to TBP (6, 22, 23), and it was difficult toimagine how TFIID can function without its primary TBPbinding site. Besides hTAFII250, interactions between TBP andthe metazoan TFIID subunits TAFII20/dTAFII30 (31, 36),hTAFII28 (37), and hTAFII18 (37) have been described; there-fore, TBP binding to other yeast TAFIIs remained a formalpossibility, but evidence has not been presented until thisstudy. We have shown that TBP binds to TAFII68/61 and thatthe region required for its binding in vitro is also necessary forthe stable association of TBP with the TFIID complex and thestability of the TFIID complex in vivo. (Fig. 3). Redundancy inthe TBP binding interface in TFIID is supported by geneticstudies that shown that the major TBP binding region ofTAFII145p is dispensable for viability and the transcription ofmost genes in vivo (22, 23).

The prevailing evidence indicates that the histone-likeTAFIIs and TAFII25 forms the scaffold, or core, of the TFIIDcomplex. This hypothesis is based on the data arising from thesystematic analysis of the requirements for histone-like TAFIIsversus other TAFIIs in transcription and preservation of TFIIDstructure. Inactivation of ts2 mutations in every histone TAFII

gene has drastic consequences on TFIID structure in vivo, asevidenced by the rapid turnover of multiple TAFIIs and TBP atthe restrictive temperature (Michel et al. (16) and this report).In striking contrast, inactivation of temperature-sensitive mu-tants of TAF145 or TSM1 or depletion of TAFII145 protein isnot as nearly detrimental to TFIID structure (9, 10). It seemsappropriate at this time to reevaluate the previously acceptedmodel that depicts TAFII145p/dTAFII250 as the scaffold of theTFIID complex. It is conceivable that in the absence ofTAFII145, such as in TAFII-depleted cells, a partially function-ing complex containing TBP and other TAFIIs is capable ofcarrying out gene regulatory functions. In fact, multiple TFIIDcomplexes are found in mammalian cells (for review, see Ref.

38). Evidence for such a complex has not been presented, and itmay be difficult to do so because of the differences in theenvironment within the cell versus cell extracts. It is becomingincreasingly clear that before we can interpret all of the dataemerging about the role of TAFIIs in transcription, multiplealleles need to be analyzed, and we must have a good under-standing of how the mutations in each particular allele affectTFIID structure.

Acknowledgments—We thank Michael Green, Richard Young,Danny Reinberg, Fred Winston, and Kevin Struhl for yeast strains andantibodies, Jizhong Zou for his assistance in constructing strainYJR375, Tony Weil for communicating his findings before publication,and our laboratory colleagues and the entire Pennsylvania State Uni-versity gene regulation community for advice and reagents.

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Joseph C. Reese, Zhengjian Zhang and Harsha KurpadIdentification of a Yeast Transcription Factor IID Subunit, TSG2/TAF48

doi: 10.1074/jbc.M001635200 originally published online April 4, 20002000, 275:17391-17398.J. Biol. Chem. 

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