The long-term objective of this proposal is to gain insight into the mechanisms of control of gene expression and the role of stress proteins in cellular metabolism. More specifically, to study the regulation of transcription and the function of the genes composing the HSP70 multigene family of Saccharomyces cerevisiae. The sequences assential and sufficient for the inducibility of HSP70 will be determined by constructing deletion and point mutations in vitro and analyzing the expression of the altered promoters in vivo. The role of HSP70 in regulating its own promoter as well as transcription of other heat shock genes will be determined. Synthesis of HSP70 has been made independent of heat shock control by linking the GAL1 promoter to the HSP70 structural gene. Preliminary results indicate that transcription of the heat shock promoter is not induced in the presence of HSP70 upon a heat shock. The observation will be extended and the effect of constitutive HSP70 synthesis on the expression of other heat shock genes determined. The sequences involved in this repression of HSP70 transcription will be delineated. Mutations in the promoter region will be constructed in vitro and the expression of the altered promoters analyzed in vivo. Insight into the regulation of the heat shock response will be gained by isolating and analyzing mutations in unlinked genes involved in the regulation of transcription of the heat shock genes. We will attempt to isolate mutants which fail to induce transcription. We will also undertake a more directed attempt to isolate mutations in proteins which interact directly with the HSP70 promoter. A down mutation in the sequences required for induction will be used as a starting point, and we will attempt to isolate mutations in other genes which can overcome the effect of the first mutation and allow increased synthesis. Point mutations in the HSP70 gene will be isolated to gain insight into the function of the inducible 70K gene (YG100). These temperature-sensitive HSP70 mutations will be isolated in order to identify genes whose protein products directly or indirectly interact with HSP70. Also, the phenotypes of the point mutants will be analyzed in an attempt to separate the regulatory activities from other activities of the protein.
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