Domain structure and protein interactions of the silent information regulator Sir3 revealed by screening a nested deletion library of protein fragments

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.