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.
King
Daniel A
DA
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115, USA.
Hall
Brian E
BE
Iwamoto
Melanie A
MA
Win
Khine Zar
KZ
Chang
Ju Fang
JF
Ellenberger
Tom
T
D001483:Base Sequence; D001665:Binding Sites; D004271:DNA, Fungal / Q000235:genetics; D019281:Dimerization; D020868:Gene Silencing; D007700:Kinetics; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D021122:Protein Subunits / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000378:metabolism; D017384:Sequence Deletion; D038281:Silent Information Regulator Proteins, Saccharomyces cerevisiae / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D004271:DNA, Fungal; D009838:Oligodeoxyribonucleotides; D021122:Protein Subunits; D011994:Recombinant Proteins; C084122:SIR3 protein, S cerevisiae; C061573:SIR4 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D038281:Silent Information Regulator Proteins, Saccharomyces cerevisiae