The goals of this research plan are to elucidate the mechanisms involved in regulating transcription of glycolytic and ribosomal RNA genes in Saccharomyces cerevisiae. Distinct cis-acting sequences which mediate positive or negative regulation of transcription of the two enolase genes (ENO1 and ENO2) and one of the glyceraldehyde-3-phosphate dehydrogenase genes (TDH3) have been mapped within the 5' flanking regions of each gene. Proteins which form stable complexes with these cis-acting regulatory sequences have been identified using DNA/protein binding assays. Genetic and biochemical studies suggest that the GCR1 gene product, which is required for efficient transcription of most glycolytic genes, interferes with the binding of a repressor-like protein to a site within the upstream activation sequences (UAS) of ENO2. The GCR1 protein and the repressor protein will be purified and extensively characterized with respect to their roles in coordinate regulation of ENO1, ENO2, and TDH3 expression. Mutant strains which are defective in the repressor gene will be isolated and characterized. Utilizing these mutant strains and the purified regulatory proteins, correlations will be made between specific DNA/protein binding events and expression of the glycolytic gene under study in cells grown on glycolytic or gluconeogenic carbon soruces. A protein which binds to the upstream repression sequences (URS) in ENO1 will also be purified. The effects of the URS binding protein on the binding of other regulatory proteins to the ENO1 UAS regions or TATAAA sequences will be investigated. Mutant strains which are defective in the URS binding protein gene will be isolated and characterized. A highly sensitive assay for detecting RNA polymerase II-dependent selective initiation of transcription in vitro will be developed. Once an in vitro transcription assay is available, it will be used in conjunction with the aforementioned binding studies and genetic analyses to further elucidate the mechanisms involved in regulating glycolytic gene expression. The sequence requirements for synthesis of 34SrRNA in vivo will be further defined using a centromere plasmid carrying an artificial 35SrRNA gene. These studies will be aimed toward establishing the transcriptional function of sequences adjacent to the 34SrRNA initiation site in the presence and absence of a spacer promoter sequence which stimulates 35SrRNA synthesis in vivo more than 10-fold. Factors required for RNA polymerase I- dependent selective transcription from the spacer promoter will be purified and used to reconstitute accurate transcription in vitro.
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