The Saccharomyces cerevisiae Srb8-Srb11 Complex Functions with the SAGA Complex during Gal4-Activated Transcription

The SAGA (Spt-Ada-Gcn5-acetyltransferase) complex of Saccharomyces cerevisiae is a large multiprotein complex that is required for the normal transcription of many genes and is conserved from yeast to humans (27, 34, 54). The SAGA complex functions in vivo as a coactivator for transcriptional activation by the Gal4 protein, playing a key role in activating genes that allow S. cerevisiae to metabolize galactose as a carbon source (9, 32). Past work has shown that upon induction by galactose, Gal4 recruits SAGA and the Spt3 component of SAGA facilitates TBP binding to the GAL1 promoter (9, 10, 12, 18, 32). Spt3 has also recently been implicated in nucleosome remodeling (59). Mutations in genes encoding structural components of SAGA, such as SPT20, cause a severe Gal− phenotype (51, 57). However, after prolonged galactose induction, spt20Δ mutants can induce GAL1 to approximately 40% of wild-type levels (E. Larschan and F. Winston, unpublished results). This result is consistent with evidence that several other factors also play roles in Gal4-mediated activation, including the general factors TATA-binding protein (TBP) and TFIIB (2, 3, 33, 38, 43, 64), Swi/Snf (61), Cti6 (47), Mediator (29, 31, 38), and Srb8-Srb11 (1, 6, 25, 30, 39). To learn more about factors involved in Gal4-mediated activation and their possible relationship to the role of SAGA, we performed a selection for suppressors of the spt20Δ Gal− phenotype and identified eight complementation groups. These studies identified one group as the SRB9 gene and led us to analyze the requirement for Srb9 in GAL1 activation. Srb9 is part of the Srb8-Srb11 complex, which has been shown to be involved in many aspects of transcription in vivo (reviewed in reference 13). Although this complex is physically associated with Mediator, it is both biochemically and genetically separable from the core Mediator (11, 13, 39). Phenotypic analysis has shown that srb8Δ-srb11Δ mutants possess many common phenotypes, including slow growth on galactose-containing medium and flocculence (6, 30, 39). Transcriptional studies have demonstrated that Srb8-Srb11 plays both positive and negative roles in transcription (13), affecting genes involved in carbon metabolism (30, 39, 56), stationary-phase entry (15), and the nitrogen starvation pathway (46). Srb10, a member of Srb8-Srb11, is a protein kinase that shares homology with cyclin-dependent kinases (CDKs), particularly mammalian CDK8 (reviewed in reference 44). One putative role for Srb10 in transcription was suggested by the demonstration that it can phosphorylate the largest subunit of RNA polymerase II on its carboxy-terminal domain on serine 2 and serine 5 in vitro (11, 30, 39). In vivo targets of the Srb10 kinase include the activators Gcn4 (17), Msn2 (17), Ste12 (46), Gal4 (25), and the Mediator component Med2 (22). Furthermore, in vitro transcription experiments have suggested both positive and negative roles for Srb10 (23, 40). A mutant of Srb10 that lacks kinase activity shares phenotypes with srb8Δ, srb9Δ, srb10Δ, and srb11Δ mutants, indicating that Srb10 kinase activity is required for Srb8-Srb11 function (39). To further understand the role of Srb8-Srb11 and SAGA during Gal4 activation, we examined the binding of Srb9 to the Gal4-activated GAL1 promoter by chromatin immunoprecipitation (ChIP). Our results suggest that Srb8-Srb11 associates with the GAL1 promoter in a SAGA-dependent fashion. Furthermore, the association is dependent on the SAGA component Spt3, but not Gcn5. We also show that Srb10 kinase activity is required for activation of GAL1 and the association of TBP with the GAL1 TATA element, consistent with previous results indicating that Srb10 kinase activity stimulates activity of reporter genes fused to the GAL1 promoter (30, 39). In contrast to SAGA, Srb8-Srb11 requires TBP binding for its association with the GAL1 promoter, indicating that these factors mutually stabilize each other at the GAL1 promoter. Taken together, these studies expand our understanding of the steps required for Gal4 activation of transcription initiation.