Analysis of General Transcription Factors in Yeast

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The long-term objectives of this application are to learn more about fundamental and conserved aspects of transcription initiation in eukaryotes. The studies will focus on a protein complex discovered in the yeast, Saccharomyces cerevisiae, that controls transcription initiation by RNA polymerase I1. This complex, named SAGA (Spt-Ada-Gcn5 Acetyltransferase), is a member of a class of factors, called coactivators, that play critical roles in the control of transcription initiation. Coactivators are large, multi- protein complexes that often possess multiple activities and in some cases can both activate and repress transcription. SAGA is conserved, as human SAGA-like complexes have been identified. Several issues about coactivators are poorly understood, including their mechanisms of activation, the functional redundancy between different coactivators, and how some coactivators can both activate and repress transcription. The proposed experiments are to study several issues concerning SAGA's functions in yeast. Specific Aim 1 contains three sets of experments. The first two sets address the mechanism of activation by the SAGA component, Spt3, using biochemical and genetic approaches. The third set addresses the possible role of acetylation of some SAGA subunits. Specific Aim 2 addresses the in vivo coordination of SAGA's activities with other coactivator complexes. The first section combines genetic analysis with microarray studies to address the functional redundancy between SAGA and a second coactivator complex, Swi/Snf. The second section is designed to identify other factors that function with SAGA to activate transcription of the well-studied GAL 1 promoter. Specific Aim 3 shifts focus to address repression of transcription. Mot3 is a DNA-binding repressor that is functionally related to SAGA. Mot3 plays a critical role in the repression of genes required for synthesis of ergosterol, a key component of S. cerevisiae membranes. Strong repression of these genes also requires low oxygen levels. Two sets of experiments are proposed. First, the mechanism of repression will be addressed, testing the preliminary finding that SAGA is also required for repression of these genes, identifying other factors necessary, and elucidating their respective roles. Second, the role of oxygen-mediated regulation will be addressed using molecular and genetic analysis. These studies should reveal important aspects of transcriptional control in yeast. Given the strong conservation of transcriptional mechanisms, the results will be applicable to understanding transcription in humans.