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首页> 外文期刊>Molecular and Cellular Biology >Phylogenetic Conservation and Homology Modeling Help Reveal a Novel Domain within the Budding Yeast Heterochromatin Protein Sir1
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Phylogenetic Conservation and Homology Modeling Help Reveal a Novel Domain within the Budding Yeast Heterochromatin Protein Sir1

机译：系统发育的保守性和同源性建模帮助揭示酵母芽异染色质蛋白Sir1内的一个新领域。

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摘要

The yeast Sir1 protein's ability to bind and silence the cryptic mating-type locus HMR>a 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 HMR>a. 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 [Sir127-149]) is similar in sequence to the Sir1OIR; homology modeling predicted a structure for Sir127-149 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 HMR>a 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.
机译：酵母Sir1蛋白结合并沉默隐性交配型基因座 HMR > a 的能力要求Sir1与起源识别复合体（ORC）之间存在蛋白质-蛋白质相互作用。 Sir1的C末端一半的区域，Sir1的ORC相互作用区域（Sir1OIR）和Orc1（ORC的最大亚基）内的保守溴邻同源性（BAH）域介导了这种相互作用。 Sir1OIR-Orc1BAH复合物的结构是已知的。 Sir1OIR和Orc1BAH在体外具有高亲和力相互作用，但是当在体内过量生产时，Sir1OIR不会抑制Sir1依赖性沉默，这表明Sir1的其他区域有助于其结合 HMR > a 。比较不同的Sir1蛋白发现在Sir1特征不佳的N末端一半内，有两个高度保守的区域N1和N2。 Sir1的N端部分（残基27至149 [Sir1 27-149 ]）的序列与Sir1OIR相似。同源性建模预测了Sir1 27-149 的结构，其中N1在Sir1上形成类似于已知Orc1BAH相互作用表面的子模块。与这些发现一致的是，两个杂交实验表明，Sir1 N末端可以与BAH结构域相互作用。 N1或N2内或附近的氨基酸取代降低了全长Sir1结合和沉默 HMR > a 以及与Orc1BAH相互作用的能力，这是一种两杂交法。纯化的重组Sir1形成了一个大的蛋白酶抗性结构，其中Sir1OIR域得到了保护，并且Orc1BAH在体外比全长Sir1更有效地结合Sir1OIR。因此，Sir1 N末端在Sir1-ORC沉默复合物的形成中显示出正作用和负作用。这种功能对偶性可能有助于Sir1对体内沉默子结合的ORC的选择性。

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