| The S. cerevisiae SAGA complex is a multifunctional coactivator complex that regulates a large number genes transcribed by RNA polymerase II. This thesis examines the structure, organization, and function of SAGA using three different approaches. First, we characterized the role of the Spt7 core component in SAGA. We showed that Spt7 is important in controlling the protein levels of the Spt20 and Ada1 structural components, thus regulating SAGA levels. Through biochemical and genetic analyses of a series of spt7 deletion mutants, we have identified a region of Spt7 required for interaction with the Spt8 subunit. An adjacent domain is required for the formation of a processed form of Spt7 that is present in the SLIK/SALSA complex. We also ascertained the interdependence of subunit associations within SAGA, establishing a basis for understanding SAGA organization. To extend our knowledge of SAGA structure, we determined the three-dimensional structure of SAGA using electron microscopy. The structure of SAGA, formed by five domains with interconnecting linker regions, is strikingly similar to that of the human TFTC complex, a human homologue of SAGA. Specific functions are localized to different domains, suggesting a modular organization for SAGA that provides insight into the assembly and potential coordination of SAGA activities. Finally, we showed that SAGA acts as a transcriptional repressor of the SER3 gene. Transcriptional analysis and chromatin immunoprecipitation studies demonstrated that SAGA regulates the expression of SRG1, a non-coding RNA in the 5' regulatory region of SER3. SAGA binding to the SRG1 promoter requires elements within the SRG1 upstream activating sequence. Moreover, SAGA controls the transcription of SRG1, specifically via the functions of the Spt3 and Spt8 subunits. Our data suggest that SAGA represses SER3 by regulating SRG1 transcription, which inhibits the binding of activators to the SER3 promoter. In conclusion, our studies elucidate the functional organization of SAGA and demonstrate a new role for SAGA in the transcriptional repression of the SER3 locus. |
