The regulatory circuit that acts upon the GAL genes of the yeast S. cerevisiae is well defined genetically. Of central interest is the mechanism by which the GAL4-encoded regulatory protein activates transcription of the GAL genes. This protein binds to sequences located upstream of each gene that is regulates; the events that occur subsequent to DNA binding that lead to activation of transcription are unknown. Also poorly understood are the factors responsible for catabolite repression of the GAL genes caused by growth on glucose. The long-term goal of the proposed research is to learn how proteins interact with each other and with the GAL control regions to regulate transcription. We will focus on three types of proteins: 1) the GAL4-encoded protein, 2) proteins that interact with the GAL4 protein, and 3) proteins responsible for catabolite repression of the GAL genes.
The specific aims are: 1) To locate functional regions of the GAL4-encoded protein by identifying mutations that affect the DNA binding, transcription activation, and other functions of this protein. Because the location of functional regions in this large protein are poorly defined, several in vivo will then be carried out on more precisely defined regions. 2) To identify proteins that interact with the GAL4 protein. The GAL4 protein likely activates transcription by interacting with proteins that act close to the transcription initiation site, but genes encoding such proteins have not been transcription initiation site, but genes encoding such proteins have not been identified. The genetic selections proposed here are aimed at identification of these genes. 3) To elucidate the mechanism of catabolite repression of the GAL genes by isolating and characterizing mutants defective in this regulatory circuit, using selection schemes specifically developed for this purpose. 4) To develop a genetic method for the analysis of DNA binding proteins. These methods will be applied to the study of GAL4 protein, as well as other DNA binding proteins.
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