The primary objective of the proposed experimental plan is to elucidate the mechanism by which a complex cis-acting element mediates activation of expression of the yeast enolase gene ENO2. The GCR1 gene encodes a positive regulatory protein which is required for high level transcription of yeast glycolytic genes including ENO2. Sequences that mediate GCR1-dependent activation of ENO2 expression have been identified. This regulatory element (GCR1-dependent UAS element) contains two binding sites for RAP1 protein and a binding site for ABF1 protein that play important roles in modulating the transcriptional activity of the regulatory element. GCR1 protein will be purified and binding sites for GCR1 protein will be mapped within the GCR1-dependent UAS element. The role of GCR1 binding, alone and in combination with ABF1 and RAP1, in modulating the activity of truncated, mutant, and full length versions of the GCR1-dependent UAS element will be tested in vivo. Dominant mutations in the SGC1 gene were previously identified as suppressors of both growth and transcriptional defects caused by a gcr1 null mutation. SGC1, encodes a member of the basic-helix-loop-helix (bHLH) family of DNA binding proteins. SGC1 bHLH protein(s) will be purified and binding sites for SGC1 bHLH protein(s) will be mapped within or adjacent to the GCR1-dependent UAS element. The role of SGC1 bHLH protein binding, alone and in combination with ABF1, RAP1, and GCR1, in modulating the activity of the GCR1-dependent UAS element will be studied in vivo to determine how these proteins act in combination to regulate activation of ENO2 expression. The mechanism whereby GCR1, SGC1, and RAP1 modulate ENO2 UAS element-dependent activation of transcription will be further studied using in vitro transcription assays and purified GCR1, SGC1, and RAP1 proteins. The transcriptional activities of two molecular forms of GCR1 protein encoded by two distinct GCR1 transcripts will be compared. A collection of gcr1 suppressor mutants will be screened to distinguish novel genes that modulate the transcriptional activity of the GCR1-dependent UAS element from ENO2. Finally, recent experiments show that the yeast SIN3 and SIN4 gene products function together to regulate the biochemical properties of REB1 and RAP1 DNA proteins. Genetic studies showed that sin3 or sin4 null mutations dramatically altered the biological activities of a RAP1-dependent UAS element, the REB1-dependent ENO1 URS element, and the REB1-dependent yeast ribosomal enhancer/terminator element. Although little is known about their mechanism of action, REB1 and RAP1 appear to be global regulators of yeast gene expression (RAP1 modulates the activity of the GCR1-dependent UAS element described above). Experiments are proposed to investigate the mechanism whereby SIN3 and SIN4 proteins regulate the activities of REB1 and RAP1. The long term goal of this investigation is to understand the mechanisms whereby multiple regulatory proteins act in concert to modulate eucaryotic gene expression.
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