SPT3 interacts with TFIID to allow normal transcription in Saccharomyces cerevisiae.

Abstract

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.

Synergistic Transcriptional Activation by TATA-Binding Protein and hTAFII28 Requires Specific Amino Acids of the hTAFII28 Histone Fold

“…SPT3 in yeast plays a role in transcription from a subset of promoters with nonconsensus TATA elements (9). Genetic experiments have shown a reciprocal allele-specific suppression of mutants in the putative ␣2-helix of yeast SPT3 (ySPT3) (E240K) and the H1Ј helix of yTBP (allele spt15-2; G174E) (9,11,20). This suggests that these two helices mediate the functional and perhaps also physical interactions between the respective proteins.…”

Human ubiquitin‐protein ligase Nedd4: expression, subcellular localization and selective interaction with ubiquitin‐conjugating enzymes

“…Recent reports implicated Nedd4 and its yeast homologues Rsp5/Npi1/Pub1 in multiple biological functions, including transcription and membrane transport. (i) Saccharomyces cerevisiae Rsp5 was originally identified as a revertant of mutations in the Spt3 gene encoding a TFIID-binding protein (Eisenmann et al 1992). Rsp5 is also required for the degradation of Gap1 and Fur4 permeases (Hein et al 1995), the endocytosis of uracil permease (Galan et al 1996), and the minichromosome maintenance (Yashiroda et al 1996).…”

Positive and negative functions of the SAGA complex mediated through interaction of Spt8 with TBP and the N-terminal domain of TFIIA

“…It has been proposed that the Spt3 subunit interacts directly with TBP (Bhaumik and Green 2001;Larschan and Winston 2001). Mutations on opposite sides of the TBP surface have been found to suppress an Spt3 mutation, and a mutation in Spt3 was found to suppress TBP mutations (Eisenmann et al 1992). Spt3 was found to coimmune precipitate with TBP, but only when overexpressed.…”

“…Spt3 was found to coimmune precipitate with TBP, but only when overexpressed. This may have been due to nonspecific interaction because Spt8, another SAGA subunit, did not coimmune precipitate with TBP (Eisenmann et al 1992(Eisenmann et al , 1994). Protein-protein interaction studies and two-hybrid analysis have not shown any direct interaction between Spt3 and TBP (Eisenmann et al 1994;Madison and Winston 1997).…”