FUNCTIONAL ANALYSIS OF TFIIB PROTEIN IN S CEREVISIAE

Children'S Hospital Med Ctr (Cincinnati), Cincinnati OH

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Abstract

DESCRIPTION: The long term goal of this project is to understand the molecular mechanisms of gene activation in eukaryotes using the general transcription factor TFIIB in the yeast, Saccharomyces cerevisiae. In previous work from this laboratory, mutations in a species-specific region of TFIIB were shown to affect the expression of a reporter gene activated by the activator HAP4, but not non-activated transcription or activation by GAL4. This provides strong support for the idea that yTFIIB participates in the process of gene activation in living cells. Three specific aims are proposed in this application. Specific Aim 1 will test the hypothesis that mutations in the species-specific region of TFIIB affect the interaction between HAP4 and TFIIB, thus weakening the ability of HAP4 to activate transcription. Both biochemical and genetic studies will be conducted to test this hypothesis directly. Specific Aim 2 will test the hypothesis that additional regions of TFIIB participate in the process of gene activation in vivo. TFIIB mutants will be sought that either fail to respond to activators or that bypass the requirement for activators. It is hoped that the characterization of these mutants will help reveal additional steps or events critical for gene activation in vivo. Specific Aim 3 will test the hypothesis that additional proteins important for transcription and gene activation can be revealed by screens for suppressors of temperature sensitive yTFIIB mutants. The isolation and characterization of such transcription factors will further determine how the TFIIB mutants affect the process of gene activation in vivo.