The goal of this research is to define the roles of separate components of the yeast transcriptional silencer at HMR E. Two components of the silencer at HMR E are binding sites for proteins that act else where as gene activators. A third components exhibits properties of an origin of DNA replication. This research will address the issue of how these components combine to create a silencer by answering the following question. Are gene activator function and DNA replication directly involved in silencing, or are these properties of the parts of the silencer incidental to its function as a whole? The approach taken in this proposal is aimed at systematically identifying the minimum constituents needed to generate a silencing effect on test minichromosomes defining the interactions of these constituents using primarily genetic techniques. The experiments described here will help characterize classes of protein and protein-protein and protein-DNA interactions generally employed to create compound regulatory elements. Due to the nature of the structure of the silencer the proposed experiments will not only yield information on mechanisms of transcriptional repression and hierarchies of chromosome organization, but will also open new avenues for addressing the function of gene activators and origins of DNA replication. Investigations of these issues in yeast will have general applicability and will accelerate the analysis of these same problems in higher eukaryotes
