The objective is to understand the regulation of a eukaryotic gene in terms of nucleic acid-protein interaction and metabolic regulation of effector molecules.
The specific aims are to characterize the DNA sequences required for regulated expression and to identify and characterize the proteins which interact with these sequences. Specific nucleotide changes in the twenty-two base pair dyad (the upstream activation site, or UAS) which mediates regulation of the alcohol dehydrogenase 2 gene of yeast will be constructed and characterized to identify potential points of contact between the DNA and ADR1, the protein which apparently interacts at the UAS. The domains of ADR1 mediating nuclear entry, DNA binding, transcriptional activation, and regulation will be identified by analysis of deletion mutants. The putative DNA binding and transcription activation domain of ADR1 will be analyzed by classical and recombinant genetics (using oligonucleotide directed mutagenesis) and by constructing hybrid regulatory genes. Characterization of a putative TATA or CAP regulator factor encoded by ADR6 will be achieved by genetic and biochemical means. A better understanding of alcohol metabolism and the genes controlling ethanol synthesis and degradation are anticipated medical benefits of these studies. A basic understanding of how proteins control gene action will ultimately be of medical benefit as well.
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