The long range goal of this project is to identify and characterize molecular mechanisms regulating gene expression in eukaryotes. We have chosen maltose fermentation in Saccharomyces as an example of a regulated system. We have chosen maltose fermentation in Saccharomyces as an example of a regulated system. The initial steps of maltose fermentation are carried out by maltose permease and maltase. Synthesis of these enzymes is induced by maltose and the Mal-activator mediates this regulation. The Ma1-activator is a DNA-binding transcription activator. We identified the functional domains of the Ma-1 activator, and localized the maltose- responsive regulatory domain to approximately the 220 residues at the C- terminus of this 470 residue protein. Our results indicate that several negative and positive regulatory sequence elements are found in the regulatory domain suggesting that several proteins interact with this region of Ma163p and regulate the transactivation domain of Ma163p in a sequence-specific manner. During the next grant period, we plan to test our model of maltose-dependent regulation of the Mal-activator. We will carry out a biochemical analysis of the Mal-activator protein and identify interacting proteins involved in maltose-regulated transcription activation. We will explore the regulatory role of subcellular localization, dimerization, formation of high molecular weight complexes, and post-translational modification in controlling DNA-binding and transcription activation by the Mal-activator. We will carry out a systematic mutation analysis of the regulatory domain of the Mal-activator to identify and localize the negative and positive regulatory sequence elements in this region. Allele-specific and high-copy suppressors of these mutant alleles will be isolated in an effort to identify proteins which interact with the Mal-activator at these sites. The Two-hybrid method will be used top identify proteins which interact with the Mal- activator. We will use molecular genetic approaches to determine whether maltose binds directly to the Ma1-activator. Mutations which broaden inducer specificity will be isolated. The alpha-methylglucoside-activator gene will be isolated and used to characterize the inducer sensing- signaling pathway.
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