9513412 Denis The goal of this research is to elucidate the mechanisms of eucaryotic gene regulation. The system chosen for study is the transcriptional activator ADR1 in its control of gene expression in the yeast Saccharomyes cerevisiae. The ADR1 protein contains four transactivation domains (TADs) and at least one inhibitory region (residues 227-229). The function of the TADs has been found to be highly dependent on the ADA2 coactivator complex. Moreover, ADA2 components interact specifically with individual ADR1 TADS, suggesting the ADA2 complex is a coactivator for ADRl. In addition, the 227-239 region of ADRl has been implicated in binding a repressor protein that inhibits ADR1 function independent of the promoter context. The central focus of this grant is to determine hmw the ADR1 activation and inhibitory domains interact with coactivators and repressors in bringing about the control of ADR1 function. The physical association of the ADA2 complex with ADRl will be explored biochemically, and the regions of the TADs required for binding this coactivator complex will be identified. The regions and residues of the ADRl TADs required for transactivation will be determined and correlated to those regions required for binding to the ADA2 complex. Using several complementary approaches, the putative repressor that binds the ADR1 inhibitory region will be cloned and characterized. These studies will help elucidate the complex interplay of a number of factors that can be involved in the control and action of transcriptional activators. %%% The focus of this project is a protein activator, ADRl, which stimulates the gene expression of certain proteins in the yeast Saccharomyces cerevisiae. ADRl binds to the DNA next to genes that produce enzymes that allow yeast to metabolize certain compounds when they are growing in air. The investigator has shown that ADR1 stimulates the production of RNA messages that produce the enzymes. ADR1 is a large protein containing four unique amino aci d segments that are required for the activation activity. His laboratory has found these segments must bind to certain other proteins to produce activation of gene expression. These other protein "coactivators" are a complex consisting of three proteins, ADA2/ADA3/GCN5. Proteins ADA2 and GCN5 specifically touch the sequences mentioned above. Another segment of ADR1 binds another protein that inhibits ADR1 activity. The investigator will characterize the contact areas between ADR1 and other proteins that are involved in regulating gene expression by ADR1. ***
