Abstract
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.
Highlights
JULY 21, 2006 VOLUME 281 NUMBER 29 lish silent DNA [8, 9]
We showed that a small patch of surface residues located in the middle of the Sir4 coiled-coil (Sir4-CC) mediates the interaction with Sir3 [27]
Isolation of Sir3 Domains Interacting with Sir4—The yeast silent information regulators Sir3 and Sir4 are large proteins that are prone to aggregate, perhaps reflecting their biological activity of spreading along chromatin to form transcriptionally silent DNA [43]
Summary
Identification of Functional Protein Domains from a Nested Deletion Expression Library: Methodology and Biases—We wished to create a large and unbiased library expressing random Sir protein fragments. The protein complex was concentrated to 10 mg/ml in S200 Buffer (50 mM Hepes (pH 7.6), 500 mM KCl, 1 mM DTT, 1 mM PMSF, and 1 mM EDTA) and divided into aliquots, flash frozen in liquid nitrogen, and stored at Ϫ80 °C until use. To confirm that Sir did not form disulfide bond-linked aggregates, two cysteines in the Sir4-CC were alkylated with iodoacetamide, after first reducing the proteins with 10 mM DTT in a degassed buffer containing 50 mM Hepes (pH 7.6), 100 mM KCl, and 1 mM EDTA and removing DTT by passing the sample over a NAP25 column (Amersham Biosciences) before alkylation for 30 min at room temperature with 10 mM iodoacetamide. The alkylated protein failed to react with dithionitrobenzoate, confirming the chemical blockage of both cysteines
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