Kimmerly W, Buchman A, Kornberg R, Rine J
Biochemistry Department, University of California, Berkeley 94720.
EMBO J. 1988 Jul;7(7):2241-53. doi: 10.1002/j.1460-2075.1988.tb03064.x.
The HMR E silencer is required for SIR-dependent transcriptional repression of the silent mating-type locus, HMR. The silencer also behaves as an origin of replication (ARS element) and allows plasmids to replicate autonomously in yeast. The replication and segregation properties of these plasmids are also dependent on the four SIR genes. We have previously characterized two DNA-binding factors in yeast extracts that recognize specific sequences at the HMR E silencer. These proteins, called ABFI (ARS-Binding Factor) and GRFI (General Regulatory Factor), are not encoded by any of the SIR genes. To investigate the biological roles of these factors, single-base-pair mutations were constructed in both binding sites at the HMR E silencer that were no longer recognized by the corresponding proteins in vitro. Our results indicate that the GRFI-binding site is required for the efficient segregation of plasmids replicated by the HMR E silencer. SIR-dependent transcriptional repression requires either an intact ABFI-binding site or GRFI-binding site, although the GRFI-binding site appears to be more important. A double-mutant silencer that binds neither ABFI nor GRFI does not mediate transcriptional repression of HMR. The replacement of HMR E with a chromosomal origin of replication (ARS1) allows partial SIR-dependent transcriptional repression of HMR, indicating a role for replication in silencer function. Together, these results suggest that the SIR proteins influence the properties of the HMR E silencer through interactions with other DNA-binding proteins.
HMR E沉默子是SIR依赖的沉默交配型基因座HMR转录抑制所必需的。该沉默子还具有复制起点（ARS元件）的功能，可使质粒在酵母中自主复制。这些质粒的复制和分离特性也依赖于四个SIR基因。我们之前已鉴定出酵母提取物中的两种DNA结合因子，它们可识别HMR E沉默子上的特定序列。这些蛋白质分别称为ABFI（ARS结合因子）和GRFI（一般调节因子），它们并非由任何一个SIR基因编码。为了研究这些因子的生物学作用，我们在HMR E沉默子的两个结合位点构建了单碱基对突变，体外实验表明相应蛋白质不再识别这些位点。我们的结果表明，GRFI结合位点对于由HMR E沉默子复制的质粒的有效分离是必需的。SIR依赖的转录抑制需要完整的ABFI结合位点或GRFI结合位点，尽管GRFI结合位点似乎更为重要。既不结合ABFI也不结合GRFI的双突变沉默子不能介导HMR的转录抑制。用染色体复制起点（ARS1）取代HMR E可使HMR实现部分SIR依赖的转录抑制，这表明复制在沉默子功能中发挥作用。总之，这些结果表明SIR蛋白通过与其他DNA结合蛋白相互作用来影响HMR E沉默子的特性。
