Mutational analysis of the Sir3 BAH domain reveals multiple points of interaction with nucleosomes.
Sir3, a component of the transcriptional silencing complex in the yeast Saccharomyces cerevisiae, has an N-terminal BAH domain that is crucial for the protein's silencing function. Previous work has shown that the N-terminal alanine residue of Sir3 (Ala2) and its acetylation play an important role in silencing. Here we show that the silencing defects of Sir3 Ala2 mutants can be suppressed by mutations in histones H3 and H4, specifically, by H3 D77N and H4 H75Y mutations. Additionally, a mutational analysis demonstrates that three separate regions of the Sir3 BAH domain are important for its role in silencing. Many of these BAH mutations also can be suppressed by the H3 D77N and H4 H75Y mutations. In agreement with the results of others, in vitro experiments show that the Sir3 BAH domain can interact with partially purified nucleosomes. The silencing-defective BAH mutants are defective for this interaction. These results, together with the previously characterized interaction between the C-terminal region of Sir3 and the histone H3/H4 tails, suggest that Sir3 utilizes multiple domains to interact with nucleosomes.
Sampath
Vinaya
V
Department of Biochemistry and Cell Biology, Stony Brook University, Stony Brook, NY 11794-5215, USA.
Yuan
Peihua
P
Wang
Isabel X
IX
Prugar
Evelyn
E
van Leeuwen
Fred
F
Sternglanz
Rolf
R
D000595:Amino Acid Sequence; D000818:Animals; D004252:DNA Mutational Analysis; D015966:Gene Expression Regulation, Fungal; D020868:Gene Silencing; D006657:Histones / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D009707:Nucleosomes / Q000378:metabolism*; D010641:Phenotype; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism; D038281:Silent Information Regulator Proteins, Saccharomyces cerevisiae / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism
D006657:Histones; D009707:Nucleosomes; D011993:Recombinant Fusion Proteins; C084122:SIR3 protein, S cerevisiae; D038281:Silent Information Regulator Proteins, Saccharomyces cerevisiae