Multiple interactions in sir protein recruitment by Rap1p at silencers andtelomeres in yeast

Initiation of transcriptional silencing at mating type loci and telomeres i n Saccharomyces cerevisiae requires the recruitment of a Sir2/3/4 (silent i nformation regulator) protein complex to the chromosome, which occurs at le ast in part through its association with the silencer- and telomere-binding protein Rap1p. Sir3p and Sir4p are structural components of silent chromat in that can self-associate, interact with each other, and bind to the amino -terminal tails of histones H3 and H4. We have identified a small region of Sir3p between amino acids 455 and 481 that is necessary and sufficient for association with the carboxyl terminus of Rap1p but not required for Sir c omplex formation or histone binding. SIR3 mutations that delete this region cause a silencing defect at HMR and telomeres. However, this impairment of repression is considerably less than that displayed by Rap1p carboxy-termi nal truncations that are defective in Sir3p binding. This difference may be explained by the ability of the Rap1p carboxyl terminus to interact indepe ndently with Sir4p, which we demonstrate by in vitro binding and two-hybrid assays. Significantly, the Rap1p-Sir4p two-hybrid interaction does not req uire Sir3p and is abolished by mutation of the carboxyl terminus of Rap1p. We propose that both Sir3p and Sir4p can directly and independently bind to Rap1p at mating type silencers and telomeres and suggest that Rap1p-mediat ed recruitment of Sir proteins operates through multiple cooperative intera ctions, at least some of which are redundant. The physical separation of th e Rap1p interaction region of Sir3p from parts of the protein required for Sir complex formation and histone binding raises the possibility that Rap1p can participate directly in the maintenance of silent chromatin through th e stabilization of Sir complex-nucleosome interactions.