Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115, USA.
Transcriptional silencing in yeast is mediated by the interactions of silent information regulator (Sir) proteins with chromatin and with one another. The stable association of Sir3 with Sir4 is mediated by a C-terminal region of Sir3 that has additional functions including the dimerization of Sir3. We have developed a simple, robust expression screening methodology that allows for the unbiased identification of functional protein domains expressed from nested-deletion libraries of full-length genes. Using these methodologies, Sir3 dimerization was shown to be mediated by two separate domains. One of these domains also binds cooperatively to the C-terminal coiled-coil motif of Sir4 and dimerization further increases the affinity of Sir3 for Sir4. The resulting Sir3-Sir4 complexes form progressively higher order assemblies with increasing protein concentration, with implications for the mechanism of gene silencing.