Gene expression is dependent upon the interaction of DNA binding factors with distinct promoter control elements to activate transcription. The expression of the HIS4 gene in yeast is under two different control systems. One of these, general amino acid control, involves a DNA binding protein, GCN4, that stimulates transcription in response to amino acid starvation by binding to TGACTC sequences in the HIS4 promoter region. A second system, which we call basal level control, stimulates HIS4 transcription in the absence of amino acid starvation. BAS1 and BAS2, two genes required for the basal level transcription of the HIS4 gene, are also required for control of purine biosynthesis. In addition, one of these genes, BAS2, is required for utilization of organic phosphates in the growth medium. Direct biochemical analysis shows that the BAS2 gene encodes a protein that binds to both the HIS4 and PHO5 promoters. The involvement of a single DNA binding protein in the regulation of histidine, adenine, and phosphate metabolism suggests that yeast may use a small number of DNA binding proteins to coordinate the regulation of diverse metabolic pathways. The goals of this project are twofold: First, to determine how HIS4 is regulated and the biological significance of this regulation in response to various physiological conditions. The second goal is to determine the precise molecular interactions that occur to achieve this regulation, including the binding of activator proteins (GCN4 and BAS2) to DNA and protein-protein interactions between activator proteins and other transcription factors. A combined genetic, molecular biological, and biochemical approach will address the following specific experimental aims: 1) Isolating the TATA binding factor and obtaining the gene specifying this factor. Studying protein-protein interactions between this factor and GCN4. 2) Isolation of the yeast nuclear factor that binds to the HIS4 promoter and competes with GCN4 for binding. Does this factor activate or repress HIS4? 3) Determine how BAS1 activates HIS4 and places HIS4 transcription under control of adenine availability. Determine how BAS1 controls adenine biosynthesis. 4) Identification of the nature of the BAS2 interaction with the HIS4 and PHO5 promoters. Determine how phosphate starvation induces HIS4 transcription. 5) Determine if HIS4 is under other basal level control mechanisms.
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