Coactivator function during Gal4-activated transcription in Saccharomyces cerevisiae

Prior to the work presented in this thesis was conducted, it was demonstrated that the SAGA complex facilitates the binding of TATA-binding protein (TBP) during transcriptional activation of the GAL1 gene of Saccharomyces cerevisiae. In this thesis, we examine whether SAGA is directly required as a coactivator in vivo using chromatin immunoprecipitation analysis. Our results demonstrate that SAGA is physically recruited in vivo to the UAS regions of the galactose-inducible GAL genes. This recruitment is dependent upon both induction by galactose and the Gal4 activation domain. A specific interaction between Spt3 and TBP in vivo is important for Gal4 transcriptional activation at a step after SAGA recruitment. These results, taken together with previous studies, demonstrate a dependent-pathway for the recruitment of TBP to GAL gene promoters consisting of the recruitment of SAGA by Gal4 and the subsequent recruitment of TBP by SAGA.; To better understand the role of SAGA and other factors during Gal4-activated transcription, we selected for suppressor mutations that bypass the requirement for SAGA and obtained eight complementation groups. We have identified three groups as the NHP10, HDA1, and SRB9 genes. Srb9 is part of the Srb8--Srb11 subcomplex of the Mediator coactivator that includes the Srb10 kinase. In addition to SAGA, Srb9 is present at the GAL1 promoter and facilitates the recruitment of TBP. Srb9 association with the GAL1 UAS requires SAGA and TBP-binding. In contrast, SAGA association with the promoter is largely independent of Srb9 and TBP. Thus, the Srb8--Srb11 complex associates with the GAL1 promoter subsequent to SAGA, and the binding of TBP and the Srb8--Srb11 complex are interdependent.; In addition, we describe studies, performed in collaboration with Don Prather, that provide evidence for a function of the TFIIS elongation factor during transcription initiation of GAL1. First, TFIIS binds the UAS of GAL1 more strongly than the coding region and is required for TBP binding to the TATA box, similar to initiation factors. TFIIS association with the GAL1 promoter requires the SAGA and Mediator complexes, and therefore it functions subsequent to these coactivators. Furthermore, TFIIS can bind to isolated Gal4-binding sites independent of TBP and RNA polymerase II binding. Yet, additional in vitro and in vivo studies are required to fully define this role.