Cubizolles Fabien, Martino Fabrizio, Perrod Severine, Gasser Susan M
Department of Molecular Biology and NCCR Frontiers in Genetics, University of Geneva, CH-1211 Geneva 4, Switzerland.
Mol Cell. 2006 Mar 17;21(6):825-36. doi: 10.1016/j.molcel.2006.02.006.
The budding yeast genome contains transcriptionally repressed domains at mating-type and telomeric loci, and within rDNA repeats. Gene silencing at telomeres requires the Silent information regulators Sir2p, Sir3p, and Sir4p, whereas only the Sir2p histone deacetylase is required for rDNA repression. To understand these silencing mechanisms biochemically, we examined the subunit structure of Sir2p-containing complexes. Sir2p alone forms a stable homotrimer, whereas the SIR complex is a heterotrimer containing one copy of each Sir protein. A point mutation in the Sir2p core domain (sir2(P394L)) compromises selectively rDNA repression. This mutation impairs homotrimerization but allows SIR heterotrimer formation. Surprisingly, when sir2(P394L) is coexpressed with wild-type Sir2p, rDNA repression increases and homotrimers form. Furthermore, coexpression of sir2(P394L) and enzymatically inactive sir2(H364Y) allows crosscomplementation of rDNA repression defects. The correlation of genetic and biochemical complementation argues that Sir2p trimerization is physiologically relevant for rDNA silencing.
芽殖酵母基因组在交配型和端粒位点以及核糖体DNA（rDNA）重复序列内含有转录抑制结构域。端粒处的基因沉默需要沉默信息调节因子Sir2p、Sir3p和Sir4p，而rDNA抑制仅需要Sir2p组蛋白脱乙酰酶。为了从生化角度理解这些沉默机制，我们研究了含有Sir2p的复合物的亚基结构。单独的Sir2p形成稳定的同三聚体，而SIR复合物是一种异三聚体，包含每种Sir蛋白的一个拷贝。Sir2p核心结构域中的一个点突变（sir2(P394L)）选择性地损害rDNA抑制。这种突变损害同三聚体化，但允许SIR异三聚体形成。令人惊讶的是，当sir2(P394L)与野生型Sir2p共表达时，rDNA抑制增强且同三聚体形成。此外，sir2(P394L)与无酶活性的sir2(H364Y)共表达可使rDNA抑制缺陷得到交叉互补。遗传互补和生化互补的相关性表明，Sir2p三聚体化在生理上与rDNA沉默相关。
