Mutations in the Saccharomyces cerevisiae gene SPT15, which encodes the TATA-binding protein TFIID, have been shown to cause pleiotropic phenotypes and to lead to changes in transcription in vivo. Here, we report the cloning and analysis of one such mutation, sptl5-21, which causes a single-amino-acid substitution in a conserved residue of TFIID. Surprisingly, the sptl5-21 mutation does not affect the stability of TFIID, its ability to bind to DNA or to support basal transcription in vitro, or the ability of an upstream activator to function in vivo. To study further the sptlS-21 defect, extragenic suppressors of this mutation were isolated and analyzed. All of the extragenic suppressors of sptlS-21 are mutations in the previously identified SPT3 gene. Suppression of sptlS-21 by these spt3 mutations is allele-specific, suggesting that TFIID and SPT3 interact and that sptl5-21 impairs this interaction in some way. Consistent with these genetic data, coimmunoprecipitation experiments demonstrate that the TFIID and SPT3 proteins are physically associated in yeast extracts. Taken together, these results suggest that SPT3 is a TFIID-associated protein, required for TFIID to function at particular promoters in vivo.[ Transcription initiation by RNA polymerase II is a complicated process requiring the formation of a multicomponent, preinitiation complex near the start site of transcription (for review, see Sawadogo and Sentenac 1990;Roeder 1991). This complex is composed of a large number of proteins, including RNA polymerase II and several general transcription factors. These general factors were originally identified by fractionation of HeLa cell nuclear extracts into a number of activities essential for the accurate initiation of transcription by RNA polymerase II in vitro (Matsui et al. 1980;Samuels et al. 1982;Davison et al. 1983). At least some of these general factors have been identified in other eukaryotic systems, including Saccharomyces cerevisiae (Buratowski et al. 1988;Cavallini et al. 1988;Hahn et al. 1989a), Drosophila melanogaster (Wampler et al. 1990), and rat (Conaway and Conaway 1991), indicating that many components of the RNA polymerase II transcription machinery have been evolutionarily conserved.One of the general factors, TFIID, is believed to begin formation of the preinitiation complex by binding to the TATA box (Sawadogo and Roeder 1985b;Reinberg et al.

A mutation in the gene that encodes Saccharomyces cerevisiae TFIID (SPT15), which was isolated in a selection for mutations that alter transcription in vivo, changes a single amino acid in a highly conserved region of the second direct repeat in TFIID. Among eight independent sptl5 mutations, seven cause this same amino acid change, Leu-205 to Phe. The mutant TFIID protein (L205F) binds with greater affinity than that of wild-type TFIID to at least two nonconsensus TATA sites in vitro, showing that the mutant protein has altered DNA binding specificity. Site-directed mutations that change Leu-205 to five different amino acids cause five different phenotypes, demonstrating the importance of this amino acid in vivo. Virtually identical phenotypes were observed when the same amino acid changes were made at the analogous position, Leu-114, in the first repeat of TFIID. Analysis of these mutations and additional mutations in the most conserved regions of the repeats, in conjunction with our DNA binding results, suggests that these regions of the repeats play equivalent roles in TFIID function, possibly in TATA box recognition.The eukaryotic general transcription factor TFIID plays a central role in transcription initiation. Binding of TFIID to the TATA box of RNA polymerase II-dependent promoters is the first step in the assembly of a complex that contains RNA polymerase II and at least four other general transcription factors (TFIIA, TFIIB, TFIIE, and TFIIF) (5,6,60). This initiation complex is sufficient to support basal levels of transcription in vitro (11,32,44,45,49,50). In addition, previous studies have shown that TFIID is important in vivo; in yeast cells, it is essential for growth and for normal transcription (7, 13).TFIID has been studied in vitro in some detail, and it has been shown to be capable of several interactions: binding to DNA, interactions with other general factors, and interactions with transcription regulatory factors (for a review, see reference 42). Since it is the first step in the assembly of the general initiation complex, binding of TFIID to DNA is thought to be a likely target for the regulation of transcription initiation. In support of this idea, several studies have suggested that TFIID interacts with a number of specific transcription activator proteins (21,22,25,30,51,56). In addition, another class of transcription regulatory proteins, variously termed coactivators, mediators, or adaptors, may be required to relay a signal from some specific transcription activators to the general initiation complex via an interaction with TFIID (3, 28, 41). Finally, other results suggest that TFIID may prevent the formation of inactive chromatin over a promoter (69). These in vitro studies indicate that activators may directly facilitate binding of TFIID to the promoter to overcome repression by nucleosomes (68,70,71).Genes that encode TFIID have been isolated from a number of species, and a comparison of the predicted amino acid sequences shows a very high degree of conservation (80 to 90% ide...

A mutation in the gene that encodes Saccharomyces cerevisiae TFIID (SPT15), which was isolated in a selection for mutations that alter transcription in vivo, changes a single amino acid in a highly conserved region of the second direct repeat in TFIID. Among eight independent spt15 mutations, seven cause this same amino acid change, Leu-205 to Phe. The mutant TFIID protein (L205F) binds with greater affinity than that of wild-type TFIID to at least two nonconsensus TATA sites in vitro, showing that the mutant protein has altered DNA binding specificity. Site-directed mutations that change Leu-205 to five different amino acids cause five different phenotypes, demonstrating the importance of this amino acid in vivo. Virtually identical phenotypes were observed when the same amino acid changes were made at the analogous position, Leu-114, in the first repeat of TFIID. Analysis of these mutations and additional mutations in the most conserved regions of the repeats, in conjunction with our DNA binding results, suggests that these regions of the repeats play equivalent roles in TFIID function, possibly in TATA box recognition.