Hickman Merrit, McCullough Kalyani, Woike Adrienne, Raducha-Grace Laura, Rozario Tania, Dula Mary Lou, Anderson Erica, Margalit Danielle, Holmes Scott G
Department of Molecular Biology and Biochemistry, Wesleyan University, Middletown, CT 06459, USA.
J Mol Biol. 2007 Apr 13;367(5):1246-57. doi: 10.1016/j.jmb.2007.01.044. Epub 2007 Jan 23.
Sir2 is a protein deacetylase that mediates transcriptional silencing at the HM loci, telomeres, and rDNA repeats in yeast. To identify functionally significant regions of the Sir2 protein, we have characterized two types of mutations. First, we used random mutagenesis to create temperature-sensitive alleles of the SIR2 gene. Mutations conferring conditional silencing can be isolated throughout the SIR2 gene, causing both enzymatic and protein interaction defects. We used external deletions to identify regions essential for silencing in the non-conserved regions of Sir2. Deletions of the Sir2 N-terminal 89 amino acid residues caused a subtle increase in silencing, while deletions encompassing residues 110-146 caused loss of Sir2 interactions with both Sir4 and Net1. This loss of protein interaction correlates with a loss of Sir2-mediated silencing, and is consistent with a model in which Net1 and Sir4 compete for interaction with Sir2. These results indicate that recognition of the binding partners of Sir2 is a key function of non-conserved sequences.
Sir2是一种蛋白质脱乙酰酶，可介导酵母中HM位点、端粒和rDNA重复序列处的转录沉默。为了鉴定Sir2蛋白的功能重要区域，我们对两种类型的突变进行了表征。首先，我们使用随机诱变来创建SIR2基因的温度敏感等位基因。赋予条件性沉默的突变可在整个SIR2基因中分离出来，导致酶促和蛋白质相互作用缺陷。我们使用外部缺失来鉴定Sir2非保守区域中沉默所必需的区域。删除Sir2 N端的89个氨基酸残基会导致沉默略有增加，而包含110 - 146位残基的缺失会导致Sir2与Sir4和Net1的相互作用丧失。这种蛋白质相互作用的丧失与Sir2介导的沉默丧失相关，并且与Net1和Sir4竞争与Sir2相互作用的模型一致。这些结果表明，识别Sir2的结合伙伴是非保守序列的关键功能。
