Molecular Characterization of Saccharomyces cerevisiae TFIID

Abstract

We previously defined Saccharomyces cerevisiae TFIID as a 15-subunit complex comprised of the TATA binding protein (TBP) and 14 distinct TBP-associated factors (TAFs). In this report we give a detailed biochemical characterization of this general transcription factor. We have shown that yeast TFIID efficiently mediates both basal and activator-dependent transcription in vitro and displays TATA box binding activity that is functionally distinct from that of TBP. Analyses of the stoichiometry of TFIID subunits indicated that several TAFs are present at more than 1 copy per TFIID complex. This conclusion was further supported by coimmunoprecipitation experiments with a systematic family of (pseudo)diploid yeast strains that expressed epitope-tagged and untagged alleles of the genes encoding TFIID subunits. Based on these data, we calculated a native molecular mass for monomeric TFIID. Purified TFIID behaved in a fashion consistent with this calculated molecular mass in both gel filtration and rate-zonal sedimentation experiments. Quite surprisingly, although the TAF subunits of TFIID cofractionated as a single complex, TBP did not comigrate with the TAFs during either gel filtration chromatography or rate-zonal sedimentation, suggesting that TBP has the ability to dynamically associate with the TFIID TAFs. The results of direct biochemical exchange experiments confirmed this hypothesis. Together, our results represent a concise molecular characterization of the general transcription factor TFIID from S. cerevisiae.The general transcription factor TFIID plays a central role in the initiation of mRNA gene transcription. TFIID is the only general transcription factor with specific TATA box binding activity, and the expression of many RNA polymerase IItranscribed genes is dependent on TFIID function (1, 26). Saccharomyces cerevisiae TFIID is composed of 15 subunits, TATA binding protein (TBP) and 14 distinct TBP-associated factors (TAFs) (61). The TBP subunit is responsible for the TATA box binding activity of TFIID, and TBP sequence and structure are both highly conserved throughout eukaryotic species. Like TBP, the TAF subunits of TFIID have also been highly conserved during eukaryotic evolution (6,22,66), and in aggregate, TAFs appear to serve multiple functions within the TFIID holocomplex. Early models, based on in vitro studies with individual recombinant subunits or subcomplexes of TFIID subunits, argued that TAFs functioned either as core promoter selectivity factors or as general coactivators, integrating signals between gene-specific trans-activating factors and the general transcription machinery (1). Although initially questioned, recent in vivo studies with S. cerevisiae and metazoans support both the coactivator and core promoter functions of TAFs within TFIID (1, 42, 59).Studies with numerous eukaryotic systems have yielded invaluable insights into the role of TFIID in the regulation of mRNA gene transcription. However, despite this large body of information regarding its functional properties, many fund...

Molecular and Genetic Characterization of a Taf1p Domain Essential for Yeast TFIID Assembly

“…We found that TFIID eluted in the same fractions in both the WT and the Ts48 strains (Fig. 5B) with elution profiles consistent with our earlier sizing analyses of TFIID (62). Thus, results from two different and independent assays, ChIP-ChIP and gel filtration, indicated that the TFIID complex in the Ts48 mutant was in fact intact even though our co-IP experiments (Fig.…”

“…Although in the co-IP experiments several Tafps, including Taf4p, were found to be dissociated from holo-TFIID in a Ts48 WCE, ChIP-ChIP assays showed that both Taf1p and Taf4p occupied a significantly large proportion of the RPS5 promoters in Ts48 cells, suggesting the presence of an intact TFIID complex in vivo. Gel filtration chromatography of WCE prepared from WT and Ts48 cells further proved that indeed there was intact TFIID holocomplex in these cells and that no partial TFIID complex existed in Ts48 extracts, consistent with our own previous published work (62,63). We conclude that, in the present study, the disruptive effects of mutations in Taf1p-region 4 were accentuated in our co-IP experiments.…”

“…These experiments showed that not all the TFIID-specific Tafps coprecipitated with Taf1p-region 4: for example, Taf1p, Taf2p, and Taf8p were not detected in these immunoprecipitates. We also note that no such dimer has yet been detected for yeast TFIID by co-IP (14,62), TFIID molecular sizing (62), or electron microscopy (39,40).…”

“…Table 1 lists the strains used in our experiments. Gel filtration chromatography of TFIID was conducted essentially as described previously (62) except that the buffer contained 150 mM NaCl. Column fractions were concentrated with trichloroacetic acid and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblot analysis to identify the elution position of native TFIID.…”

“…These data are consistent with the hypothesis that Taf1p subserves a critical scaffold function within the TFIID complex. The significance of these data with regard to TFIID structure and function is discussed.TFIID, one of several multiprotein general transcription factors required for RNA polymerase II (PolII)-catalyzed mRNA gene transcription, is comprised of the TATA-binding protein (TBP) and 14 distinct TBP-associated factors (TAFs or Tafps) that range in M r from 17,000 to 150,000 in Saccharomyces cerevisiae (62,63,65,75). These TFIID subunits share significant sequence conservation with their metazoan orthologs (1,22,26).…”