In the yeast S. cerevisiae, the alpha 1 protein is responsible for expression of a set of cell-type specific genes, the alpha-specific genes. Three such genes, MF(alpha)1, and MF(alpha)2, and STE3, have been identified and code for proteins required for the specialized functions of the alpha cell type. Our goal is to understand the molecular basis for two aspect of the alpha 1 protein's regulatory activity: 1) the specificity of alpha 1 protein for the alpha-specific genes and 2) the mechanism by which alpha 1 protein implements its activation of gene expression. We will use the results of our analysis of the action of the alpha 1 protein on the MF(alpha)1, promoter to understand how the separate elements of a yeast promoter are coordinated. To analyze the activity of the alpha 1 protein, we will begin by purifying the alpha 1 protein. We will test the idea that the alpha 1 protein is specific for activating expression of the MF(alpha)1 gene because it binds to sequences near this gene. If so, is it solely a property of alpha 1 protein or are other yeast proteins involved? Since the promoter is a multi-component system, it seems likely that alpha 1 protein exerts its effects by interacting with other transcription components; by identifying and characterizing these interacting proteins we may learn whether or not proteins that bind to separate promoter elements interact with each other. To understand how the separate sequence elements (and their associated proteins) are coordinated in promoter function, we will identify other proteins that interact with the MF(alpha)1 promoter. We will drive formation of a transcription complex with the DNA binding proteins and alpha 1 protein and will attempt to reproduce specific alpha 1 dependent transcription in vitro. In addition to our interest in how the alpha 1 protein acts, we are also interested in how the alpha 1 protein is regulated. Some strains of yeast can switch mating type frequently; the rapid acquisition of specialized cell function by the newly switched cell is suggestive evidence that the old cell type regulators, i.e., alpha 1 (and others) are rapidly inactivated after the cell type switch occurs. We propose to examine the inactivation of the alpha 1 protein to discern how this putative activity is regulated.
