title: Phylogenetic conservation and homology modeling help reveal a novel domain within the budding yeast heterochromatin protein Sir1.
abstract: The yeast Sir1 protein's ability to bind and silence the cryptic mating-type locus HMRa requires a protein-protein interaction between Sir1 and the origin recognition complex (ORC). A domain within the C-terminal half of Sir1, the Sir1 ORC interaction region (Sir1OIR), and the conserved bromo-adjacent homology (BAH) domain within Orc1, the largest subunit of ORC, mediate this interaction. The structure of the Sir1OIR-Orc1BAH complex is known. Sir1OIR and Orc1BAH interacted with a high affinity in vitro, but the Sir1OIR did not inhibit Sir1-dependent silencing when overproduced in vivo, suggesting that other regions of Sir1 helped it bind HMRa. Comparisons of diverged Sir1 proteins revealed two highly conserved regions, N1 and N2, within Sir1's poorly characterized N-terminal half. An N-terminal portion of Sir1 (residues 27 to 149 [Sir1(27-149)]) is similar in sequence to the Sir1OIR; homology modeling predicted a structure for Sir1(27-149) in which N1 formed a submodule similar to the known Orc1BAH-interacting surface on Sir1. Consistent with these findings, two-hybrid assays indicated that the Sir1 N terminus could interact with BAH domains. Amino acid substitutions within or near N1 or N2 reduced full-length Sir1's ability to bind and silence HMRa and to interact with Orc1BAH in a two-hybrid assay. Purified recombinant Sir1 formed a large protease-resistant structure within which the Sir1OIR domain was protected, and Orc1BAH bound Sir1OIR more efficiently than full-length Sir1 in vitro. Thus, the Sir1 N terminus exhibited both positive and negative roles in the formation of a Sir1-ORC silencing complex. This functional duality might contribute to Sir1's selectivity for silencer-bound ORCs in vivo.
authors.affiliation: Department of Biomolecular Chemistry, University of Wisconsin School of Medicine andPublic Health, Madison, Wisconsin 53706-1532, USA. cfox@wisc.edu
authors.forename: Zhonggang
authors.initials: Z
authors.lastname: Hou
authors.forename: John R
authors.initials: JR
authors.lastname: Danzer
authors.forename: Liza
authors.initials: L
authors.lastname: Mendoza
authors.forename: Melissa E
authors.initials: ME
authors.lastname: Bose
authors.forename: Ulrika
authors.initials: U
authors.lastname: Müller
authors.forename: Barry
authors.initials: B
authors.lastname: Williams
authors.forename: Catherine A
authors.initials: CA
authors.lastname: Fox
mesh_terms: D000483:Alleles; D000595:Amino Acid Sequence; D017124:Conserved Sequence; D020868:Gene Silencing; D006570:Heterochromatin / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D051738:Origin Recognition Complex / Q000378:metabolism; D010802:Phylogeny; D011485:Protein Binding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D037941:Silencer Elements, Transcriptional / Q000235:genetics; D038281:Silent Information Regulator Proteins, Saccharomyces cerevisiae / Q000737:chemistry* / Q000378:metabolism; D040681:Structural Homology, Protein; D020798:Two-Hybrid System Techniques
chemical_list: D006570:Heterochromatin; C497214:ORC1 protein, S cerevisiae; D051738:Origin Recognition Complex; C084121:SIR1 protein, S cerevisiae; C084122:SIR3 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D038281:Silent Information Regulator Proteins, Saccharomyces cerevisiae