The longterm objective of our work is to understand how, in eukaryotes, regulatory proteins bind to specific DNA sequences and turn genes on and off. Our experiments focus on the yeast activator GAL4 that binds to the so called galactose upstream sequence (UASG) and turns on transcription of flanking genes at distance spanning many hundreds of base pairs. This activator also turns on gene expression in mouse cells in tissue culture, and understanding the mechanism of action of GAL4 may provide us with very general insights into the molecular basis of gene regulation. We will continue our characterization of the activating surfaces of yeast transcriptional activators: we will isolate mutants affecting the activating region(s) of GAL4, design synthetic activating regions and attempt to isolate new classes of activating regions that stimulate genes not activated by GAL4. We will study the effects of yeast activatorS in mammalian and Drosophila cells. we will study the basis of the DNA binding specificity of GAL4: we will isolate mutants with altered or relaxed specificity for various defined mutant operators and test whether the """"""""cysteine fIngers of GAL4 and PPRl are responsible for sequence recognition by studying hybrids of these two molecules. We will define the part(s) of GAL4 that mediate(s) cooperative binding of GAL4 to multiple sites in vivo. We will attempt to reproduce cooperative binding of GAL4 to reiterated sites in vitro. We will study the activities of various GAL4-derived activators on constructs that vary the distance of the GAL4 binding site from the transcriptional start site. We will identify extragenic suppressors of GAL4 mutants deficient in the activation function; these suppressors might define the transcription factor(s) (e.g. RNA polymerase?) that interact with the activation function of GAL4. We will characterize the mechanism(s) by which growth in glucose blocks GAL4's stimulatory activity. We will study negative control at a distance by placing the GAL1 promoter under control of the HMR silencer and analyzing the effect on GAL4 binding and activity in vivo.
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