The long term goal of this grant is to further the understanding of how eukaryotic transcriptional regulators bind to DNA and affect gene transcription. Although the proposed experiments involve transcriptional regulators from a variety of organisms and address a variety of questions, all involve, at least in part, manipulations in yeast to facilitate achieving the goals.
The specific aims fall into three broad categories. First, a series of experiments concern the mechanism by which GAL4, a widely studied """"""""universal"""""""" activator from yeast, activates transcription. Particular attention will be paid to the effect of GAL11 an auxiliary factor that somehow helps GAL4 and certain other activators. Genetic and physiological experiments will probe the idea that GAL11 forms a complex with these activators, a complex that may involve additional proteins as well. Second, the mechanism by which members of the GAL4 family of activators recognize specific sites in DNA will be analyzed. This is a particularly intriguing example of protein DNA interaction: each of these proteins (eg GAL4, PUT3 and PPR1) bears similar """"""""zinc cluster"""""""" elements that contact identical DNA triplets; the distinguishing feature of each DNA site is evidently the spacing between these recognized triplets. Among other experiments, chimeric proteins will be analyzed to reveal the basis of the protein DNA specificity. Third, the action of the Drosophila morphogen Dorsal will be analyzed. The putative co-factor that presumably converts Dorsal from an activator into a negative regulator will be cloned, as will the natural Drosophila activator that is inhibited by Dorsal working in conjunction with its putative co-factor. The structural basis for the interaction between Dorsal and Cactus, and between Dorsal (a member of the rel class of activators) and DNA will be analyzed.
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