Gene expression in eukaryotes is often under the control of multiple nuclear proteins that work in combination to regulate transcription. The combined interactions between these proteins will often create a new form of regulatory activity that is distinctly different than the function of either protein alone. The protein-protein interactions may dictate whether a regulatory protein functions as a transcriptional activator or repressor, as well as determine which genes are regulated by the protein. We are interested in how the combined interactions between these transcriptional regulatory proteins alter their function and site of action. We have chosen to study this problem using a simple regulatory system that determines cell type in the yeast Saccharomyces cerevisiae. This system involves the alpha2 and a1 proteins, members of the highly conserved homeodomain family of DNA-binding proteins; and MCM1, a protein with strong sequence similarity to the DNA-binding domain of the mammalian serum response factor, SRF. The alpha2 protein works in combination with MCM1 to repress one set of genes, and with the a1 to repress a different set of genes. Interactions between alpha2 and MCM1, and alpha2 and a1 therefore determine the target specificity of alpha2 repression. We are interested in how these protein-protein interactions effect DNA-binding affinity and specificity of alpha2. We propose to: (1) Examine the mechanism of DNA binding by the alpha2 homeodomain. This analysis may indicate whether there are any general principals that can be applied to DNA recognition by homeodomain proteins in higher eukaryotes. (2) Determine how MCM1 binds DNA. These experiments will provide knowledge about how members of this growing class of DNA-binding proteins function. (3) Determine which residues in alpha2 and MCM1 are involved in protein-protein interactions between the two proteins, and how these interactions effect the mechanism of DNA-binding by each protein. (4) Examine the mechanism of DNA-binding by alpha2 when it is associated with the a1 protein. The proteins in this simple regulatory system are similar in structure to regulatory proteins in mammals and plants. It therefore seems likely that the information we learn about the interactions between these classes of proteins will be relevant to more complex systems in higher eukaryotes.