Central to efforts to apply genetics and genetic engineering technologies to health-related problems is our understanding of transcriptional regulation in eukaryotes. The long-range goal of the research proposed is to discover the basic molecular mechanisms that modulate gene expression in eukaryotes. The system chosen for this study is the galactose regulon of yeast because it offers the most powerful combination of genetic and biochemical approaches. Three questions of basic interest in the study of all eukaryotes will be addressed: 1) how do positive regulatory proteins bind specific DNA control regions? 2) how is negative regulation effected? 3) what are the factors involved in the transcriptional apparatus and how do they work? In the galactose regulon activation of transcription of the structural genes requires the positive regulator encoded by GAL4 gene. Only the first 74 amino acids of GAL4 protein are required for specific DNA binding but how this binding takes place is unknown. Regions of this binding domain in GAL4 will be exchanged with corresponding regions in other positive regulatory proteins to determine which sequences are involved in specific and general binding. Mutations in dysfunctional hybrid proteins which restore function will be selected. Coupled with DNA protection studies, these two approaches will provide a picture of how specific binding of DNA takes place. In the galactose regulon negative regulation is mediated by the GAL80 protein. Using in vitro produced GAL80 protein and gel- retardation assays, the interaction between GAL4 and GAL80 proteins and DNA will be investigated. The true-inducer which alters GAL4-GAL80 interaction will be determined. The characterization of the kinetics of the GAL4-GAL80 association will provide insight into the molecular mechanisms of this type of negative regulation. Upon induction GAL4 protein presumably interacts with some unknown factors which constitute the transcriptional complex. A combined genetic and biochemical approach will be used to isolate the genes and the proteins they encoded which participate initiating transcription. Mutations in genes which suppress lesions in a region of GAL4 required for transcriptional activation have been isolated. These genes will be studied further. Proteins which bind GAL4 protein will be isolated.
The aim i s to assemble all the factors necessary to reconstitute an in vitro transcription system.
