The long range goal of this project is to identify and characterize molecular mechanisms regulating gene expression in eukaryotes. Maltose fermentation in the Saccharomyces yeasts has been chosen as a model regulated system. The initial steps of maltose fermentation are carried out by maltose permease (the transport protein) and maltase (the cleavage enzyme). Synthesis of these enzymes is induced by maltose and repressed by glucose and both regulatory processes are controlled at the level of transcription. Our studies have identified a trans-acting positive factor, the MAL-activator protein, which mediates the transcriptional regulation of the structural genes encoding maltose permease and maltase. Maltose fermenting Saccharomyces strains contain at least one of a series of complex loci (MAL1, MAL2, MAL3, MAL4 and MAL6) each of which encodes maltose permease, maltase and the MAL-activator. The experiments described in this proposal are designed to reveal the details of the role played by the MAL-activator in regulating expression of the MAL structural genes. For this, we will a) complete our analysis of the functional domains of the MAL-activator using noninducible and constitutive mutations by in vitro and in vivo mutagenesis techniques; b) identify proteins which interact with the MAL-activator and play a role in maltose induction using the """"""""two hybrid protein system"""""""" and c) characterize the role of phosphorylation and/or proteolytic degradation of the MAL-activator in the regulation of fermentation. We will investigate the factors involved in regulating the synthesis of the MAL-activator itself by identifying upstream controlling elements of the MAL-activator gene. We will explore the role of MIG1 protein in the glucose repression of the MAL gene, both structural and regulatory. Finally, we plan to reinvestigate the reported position effect controlling the timed synthesis of maltase during the yeast cell cycle.
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