The general transcription factor TFIID is a multisubunit complex of TATA-binding protein (TBP) and 14 distinct TBP-associated factors (TAFs). Although TFIID constituents are required for transcription initiation of most mRNA encoding genes, the mechanism of TFIID action remains unclear. To gain insight into TFIID function, we sought to generate a proteomic catalogue of proteins specifically interacting with TFIID subunits. Toward this end, TFIID was systematically immunopurified by using polyclonal antibodies directed against each subunit, and the constellation of TBP-and TAF-associated proteins was directly identified by coupled multidimensional liquid chromatography and tandem mass spectrometry. A number of novel protein-protein associations were observed, and several were characterized in detail. These interactions include association between TBP and the RSC chromatin remodeling complex, the TAF17p-dependent association of the Swi6p transactivator protein with TFIID, and the identification of three novel subunits of the SAGA acetyltransferase complex, including a putative ubiquitin-specific protease component. Our results provide important new insights into the mechanisms of mRNA gene transcription and demonstrate the feasibility of constructing a complete proteomic interaction map of the eukaryotic transcription apparatus.

The transcription factor TFIID, a central component of the eukaryotic RNA polymerase II (Pol II) transcription apparatus, comprises the TATA-binding protein (TBP) and approximately ten TBP-associated factors (TAFs). Although the essential role of TBP in all eukaryotic transcription has been extensively analysed in vivo and in vitro, the function of the TAFs is less clear. In vitro, TAFs are dispensable for basal transcription but are required for the response to activators. In addition, specific TAFs may act as molecular bridges between particular activators and the general transcription machinery. In vivo, TAFS are required for yeast and mammalian cell growth, but little is known about their specific transcriptional functions. Using conditional alleles created by a new double-shutoff method, we show here that TAF depletion in yeast cells can reduce transcription from some promoters lacking conventional TATA elements. However, TAF depletion has surprisingly little effect on transcriptional enhancement by several activators, indicating that TAFs are not generally required for transcriptional activation in yeast.

The TATA sequence-binding factor TFIID plays a central role both in promoter activation by RNA polymerase II and other common initiation factors, and in promoter regulation by gene-specific factors. The sequence of yeast TFIID, which seems to be encoded by a single gene, contains interesting structural motifs that are possibly involved in these functions, and is similar to sequences of bacterial sigma factors.

The insulin gene is expressed almost exclusively in pancreatic S-cells. The DNA sequences that control cell-specific expression are located upstream of the transcription initiation site. To identify the cis-acting transcriptional control regions within the rat insulin II gene that are responsible for this tissue-specific expression pattern, we constructed a series of 5'-flanking deletion mutants and analyzed their expression in vivo in transfected insulin-producing and -nonproducing cell lines. Pancreatic ,8-cell-specific expression was shown to be controlled by enhancer sequences lying between nucleotides -342 and -91 relative to the transcription start site. The rat insulin II enhancer appears to be a chimera, composed of a number of distinct cis-acting DNA elements. Both positive and negative transcriptional regulatory elements appear to be responsible for this cell-type-specific expression. We have shown that expression from one element within the enhancer, which is found between nucleotides -100 and -91, is regulated by both positive-and negative-acting cellular transcription factors. Expression from chimeras containing only the enhancer element sequences from -100 to -91 were active only in insulin-producing cells, indicating that the positive-acting factor(s) required for this activity may be active only in I8-cells. In contrast to the enhancer region, the rat insulin II gene promoter did not appear to require cell-specific transcription factors. Promoter mutants with 5'-flanking sequences extending to nucleotides -90 and -73 were constitutively active in both insulin-producing and -nonproducing cells. These results suggest that rat insulin II gene transcription in pancreatic I-cells is imparted by a combination of both negative-and positive-acting cellular factors interacting with the gene enhancer.

In vivo studies have previously shown that Saccharomyces cerevisiae ribosomal protein (RP) gene expression is controlled by the transcription factor repressor activator protein 1 (Rap1p) in a TFIID-dependent fashion. Here we have tested the hypothesis that yeast TFIID serves as a coactivator for RP gene transcription by directly interacting with Rap1p. We have found that purified recombinant Rap1p specifically interacts with purified TFIID in pull-down assays, and we have mapped the domains of Rap1p and subunits of TFIID responsible. In vitro transcription of a UAS RAP1 enhancer-driven reporter gene requires both Rap1p and TFIID and is independent of the Fhl1p-Ifh1p coregulator. UAS RAP1 enhancer-driven transactivation in extracts depleted of both Rap1p and TFIID is efficiently rescued by addition of physiological amounts of these two purified factors but not TATA-binding protein. We conclude that Rap1p and TFIID directly interact and that this interaction contributes importantly to RP gene transcription.Eukaryotic mRNA gene transcription is controlled by the action of modular enhancer-binding transactivators, proteins composed of separable DNA binding domains (DBD) and activation domains (AD). DNA-bound transactivators functionally interact with the mRNA gene transcription machinery, the so-called general transcription factors (GTFs) TFIIA, -B, -D, -E, -F, and -H plus RNA polymerase II (RNAP II), to stimulate formation and/or function of the RNAP II preinitiation complex (PIC) (67). Activators also collaborate with one or more factors termed coactivators, proteins that serve as receptors for the transfactor-AD activation signal. Coactivators link transactivator-enhancer DNA binding to the PIC (43) and can be divided into several classes: those that are chromatin active, the mediator complex, and the individual components of the mRNA gene transcription machinery itself.One of the first and likely rate-limiting steps in mRNA gene transcription is the binding of TFIID to the promoter (36,40,47). TFIID is a multisubunit assembly composed of 15 evolutionarily conserved (86) subunits, the TATA-binding protein (TBP) and 14 TBP-associated factors (TAFs) (72). This GTF displays high-affinity, sequence-specific promoter DNA binding activity. Two classes of yeast mRNA-encoding genes have been defined with respect to TFIID TAF function; the first, which is TAF dependent (TAF dep ), requires TAF function for transcription, while the second and smaller group is TAF independent (TAF ind ) (29,31,45,58,81,92). Both types of genes require TBP for wild-type (WT) levels of transcription, but in the case of the TAF ind genes, TBP appears to be recruited via mechanisms distinct from TFIID (4, 80).When mRNA-encoding genes are monitored for TBP and TAF occupancy by chromatin immunoprecipitation (ChIP) assay, TAF dep genes exhibit higher TAF/TBP occupancy ratios than TAF ind genes (39, 45). Three models for TAF function have been proposed (22, 23): TAFs mediate core promoter recognition; TAFs provide essential catalytic activities tha...