The objective is to understand the regulation of a eukaryotic gene in terms of nucleic acid-protein interactions and metabolic regulation of effector molecules. More specifically we will determine the protein and DNA components that impart specificity and high affinity to zinc finger protein-DNA interactions. This will be achieved by analyzing the DNA binding properties of mutant proteins containing single amino acid changes in the two zinc fingers of ADR1. Mutants which show a change of specificity when tested with mutated binding sites will allow predictions to be made that will guide the construction of new zinc finger proteins with unique DNA binding properties. By varying the number of identical fingers in similar proteins the contribution each finger makes to the energy of binding can be calculated. Whether, and how, protein kinase A phosphorylation of ADR1 influences its transcription activation function will be determined in a variety of ways. For example, the sites and stoichiometry of phosphorylation of ADR1 will be determined and the ability of phosphorylated and nonphosphorylated ADR1 to stimulate transcription in vitro will be measured. The ADR1-independent pathway of ADH2 activation will be characterized by identifying mutants that interact uniquely through this pathway. Further analysis of ADR1-dependent mutants that alleviate glucose repression of ADH2 will focus on mutants that might reveal interactions between the ADR1-dependent and ADR1-independent pathways of gene activation and repression.. By creating new DNA binding protein with predictable binding properties it might be possible to alter the level of gene expression in desirable ways. Since several human diseases have been identified that are due to mutations in zinc finger transcription factors, understanding how these factors function is useful to an understanding of the defect in these diseases.
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