The control of gene expression underlies the development and maintenance of different cell types in eukaryotes, and often results from the complex interaction of different regulatory elements. An understanding of the molecular mechanisms of gene control will require analysis of systems in which the relevant cis- acting DNA elements and the trans-acting protein factors can be readily identified, isolated, and manipulated.
The aim of this proposal is to understand the mechanism of action of transcriptional 'silencers', novel regulatory elements which play a key role in controlling mating type in the yeast Saccharomyces cerevisiae. Yeast mating type (either a or alpha) is determined by genes at the MAT locus. Additional complete copies of mating-type genes are present at other loci (HML and HMR), but are not transcribed. These 'silent' loci act as donors of information in a mating-type switching process. The HML and HMR loci are repressed by flanking DNA sequences called 'silencers' which act over large distances (greater than 1 kb) to control transcription and transposition. The silencers contain multiple regulatory elements, and proteins which bind to two of these elements have been identified. One protein (RAP1) has been purified and its gene has been cloned. Paradoxically, RAP1 is also involved, in other contexts, in transcriptional activation. The specific (SBF-B) is involved both in silence function and DNA replication initiation. The other protein aims of this proposal are first to study the function of RAP1 by (1) mutation of the cloned gene, and (2) analysis of interacting molecules by both direct biochemical methods and by the isolation of suppressors of rap1 mutations. Other silencer binding factors (e.g. SBF-B) will be purified and cloned, and possible interactions between silencer binding factors will be examined biochemically. Finally, the DNA elements necessary and sufficient for silencer function will be determined by deletion analysis and reconstruction experiments.
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