Gardner K A, Rine J, Fox C A
Department of Biomolecular Chemistry, University of Wisconsin, Madison, Wisconsin 53706, USA.
Genetics. 1999 Jan;151(1):31-44. doi: 10.1093/genetics/151.1.31.
Silencing of the cryptic mating-type loci HMR and HML requires the recognition of DNA sequence elements called silencers by the Sir1p, one of four proteins dedicated to the assembly of silenced chromatin in Saccharomyces cerevisiae. The Sir1p is thought to recognize silencers indirectly through interactions with proteins that bind the silencer DNA directly, such as the origin recognition complex (ORC). Eight recessive alleles of SIR1 were discovered that encode mutant Sir1 proteins specifically defective in their ability to recognize the HMR-E silencer. The eight missense mutations all map within a 17-amino-acid segment of Sir1p, and this segment was also required for Sir1p's interaction with Orc1p. The mutant Sir1 proteins could function in silencing if tethered to a silencer directly through a heterologous DNA-binding domain. Thus the amino acids identified are required for Sir1 protein's recognition of the HMR-E silencer and interaction with Orc1p, but not for its ability to function in silencing per se. The approach used to find these mutations may be applicable to defining interaction surfaces on proteins involved in other processes that require the assembly of macromolecular complexes.
隐蔽交配型基因座HMR和HML的沉默需要Sir1p识别被称为沉默子的DNA序列元件,Sir1p是酿酒酵母中致力于组装沉默染色质的四种蛋白质之一。人们认为Sir1p通过与直接结合沉默子DNA的蛋白质(如起源识别复合物(ORC))相互作用来间接识别沉默子。发现了SIR1的八个隐性等位基因,它们编码在识别HMR-E沉默子能力上存在特异性缺陷的突变型Sir1蛋白。这八个错义突变都位于Sir1p的一个17个氨基酸的片段内,并且该片段也是Sir1p与Orc1p相互作用所必需的。如果通过异源DNA结合结构域直接将突变型Sir1蛋白 tethered 到沉默子上,它们就能在沉默过程中发挥作用。因此,所鉴定的氨基酸是Sir1蛋白识别HMR-E沉默子和与Orc1p相互作用所必需的,但不是其本身在沉默过程中发挥作用所必需的。用于找到这些突变的方法可能适用于定义参与其他需要组装大分子复合物的过程的蛋白质上的相互作用表面。