TAF-like function of SV40 large T antigen.

Abstract

The simian virus 40 (SV40) early gene product large T antigen promiscuously activates simple promoters containing a TATA box or initiator element and at least one upstream transcription factor-binding site. Previous studies have suggested that promoter activation requires that large T antigen interacts with both the basal transcription complex and the upstream-bound factor. This mechanism of activation is similar to that proposed for TBP-associated factors (TAFs). We report genetic and biochemical evidence suggesting that large T antigen performs a TAF-like function. In the ts13 cell line, large T antigen can rescue the temperature-sensitive (ts) defect in TAF(II)250. In contrast, neither E1a, small t antigen, nor mutants of large T antigen defective in transcriptional activation were able to rescue the ts defect. These data suggest that transcriptional activation by large T antigen is attributable, at least in part, to an ability to augment or replace a function of TAF(II)250. In addition, we show that large T antigen interacts in vitro with the Drosophila TAFs (dTAFs) dTAF(II)150, dTAF(II)110, and dTAF(II)40, as well as TBP. The relevance of these in vitro results was established in coimmunoprecipitation experiments using extracts of SV40-infected alpha3 cells that express an epitope-tagged TBP. Large T antigen was coimmunoprecipitated by antibodies to epitope-tagged TBP, endogenous TBP, hTAF(II)100, hTAF(II)130, and hTAF(II)250, under conditions where holo-TFIID would be precipitated. In addition, large T antigen copurified and coimmunoprecipitated with phosphocellulose-purified TFIID from SV40-infected alpha3 cells. Large T antigen also coprecipitated with anti-TBP antibody from extracts of ts13 cells expressing wild-type large T antigen under conditions where the ts defect in TAF(II)250 was rescued. In contrast, a transactivation mutant of large T antigen, which was unable to rescue the ts defect, failed to coprecipitate. We conclude from these data that transcriptional activation of many promoters by large T antigen results from its performing a TAF-like function in a complex with TFIID.