The yeast galactose/melibiose regulon of S.cerevisiae is being studied as a model for transcription regulation in eukaryotes. In this system GAL4 protein dependent transcription is affected by an auxillary activator, GAL11, and two types of repression; repression due to the GAL80 protein, and repression due glucose catabolism. Galactose together with GAL3 protein triggers a rapid release from GAL80 repression but not from glucose repression. The goal is to determine how the galactose and glucose signaling systems and the GAL80 and GAL11 activities affect transcription. A major thrust will be to follow up on our recent discovery that the GAL4 protein undergoes regulated phosphorylation and dephosphorylation, and to test the hypothesis that such phosphorylation state changes are key to regulating the activity state of GAL4. Several highly interrelated lines are proposed. One line is to determine if GAL4 phosphorylation alters its DNA binding and/or transcription activation functions. Mutant GAL4 proteins having altered phosphorylation sites will be subjected to in vivo and in vitro assays to establish regulatory significance of the alterations. In another line, extracts from wildtype and mutant cells grown in noninducing, inducing, or repressing media will be challenged with GAL4 and GAL80 antisera to test in vivo-responsive GAL4/GAL80 association and dissociation predicted by the current model. The question here is: how does 80 block 4 activity? A third line is to determine the mechanisms whereby galactose and GAL3 trigger the relief of the 80 block. GAL3 and other possible proteins acting early in the induction will be assayed. A fourth line is to determine precisely how in response to carbon signals the GAL3, GAL80, and GAL11 proteins and the carbon catabolite response proteins modulate the physical state of GAL4. GAL4, GAL3, GAL80 and GAL11 antibodies will be used together with in vivo and in vitro assays to detect physical associations and activities. In closely related experiments the phosphorylation state of GAL4 from hxk2, snf1, snn6, gal82, and gal83 mutants will be determined using GAL4 antibody, electrophoresis, and phospho-a.a/peptide analyses. Within the overall context of these lines, a variety of genetic and molecular experiments will be focused on identifying the proteins that act directly on, or directly in concert with GAL4 protein to bring about carbon responsive alterations in GAL4 protein dependent transcription of gal/mel genes. The proposed research will generate new information on signal transduction, regulatory ciruitry, and mechanisms operative in this system. Such information will likely have conceptual relevance to other transcriptional regulatory systems including those in human cells.
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