Gailus-Durner V, Chintamaneni C, Wilson R, Brill S J, Vershon A K
Waksman Institute of Microbiology, Rutgers University, Piscataway, New Jersey.
Mol Cell Biol. 1997 Jul;17(7):3536-46. doi: 10.1128/MCB.17.7.3536.
URS1 is a transcriptional repressor site found in the promoters of a wide variety of yeast genes that are induced under stress conditions. In the context of meiotic promoters, URS1 sites act as repressor sequences during mitosis and function as activator sites during meiosis. We have investigated the sequence requirements of the URS1 site of the meiosis-specific HOP1 gene (URS1H) and have found differences compared with a URS1 site from a nonmeiotic gene. We have also observed that the sequence specificity for meiotic activation at this site differs from that for mitotic repression. Base pairs flanking the conserved core sequence enhance meiotic induction but are not required for mitotic repression of HOP1. Electrophoretic mobility shift assays of mitotic and meiotic cell extracts show a complex pattern of DNA-protein complexes, suggesting that several different protein factors bind specifically to the site. We have determined that one of the complexes of URS1H is formed by replication protein A (RPA). Although RPA binds to the double-stranded URS1H site in vitro, it has much higher affinity for single-stranded than for double-stranded URS1H, and one-hybrid assays suggest that RPA does not bind to this site at detectable levels in vivo. In addition, conditional-lethal mutations in RPA were found to have no effect on URS1H-mediated repression. These results suggest that although RPA binds to URS1H in vitro, it does not appear to have a functional role in transcriptional repression through this site in vivo.
URS1是在多种酵母基因启动子中发现的转录抑制位点，这些基因在应激条件下被诱导。在减数分裂启动子的背景下，URS1位点在有丝分裂期间作为抑制序列起作用，而在减数分裂期间作为激活位点起作用。我们研究了减数分裂特异性HOP1基因的URS1位点（URS1H）的序列要求，并发现与非减数分裂基因的URS1位点存在差异。我们还观察到该位点减数分裂激活的序列特异性与有丝分裂抑制的序列特异性不同。保守核心序列两侧的碱基对增强了减数分裂诱导，但不是HOP1有丝分裂抑制所必需的。有丝分裂和减数分裂细胞提取物的电泳迁移率变动分析显示出DNA-蛋白质复合物的复杂模式，表明几种不同的蛋白质因子特异性结合到该位点。我们确定URS1H的一种复合物是由复制蛋白A（RPA）形成的。尽管RPA在体外结合双链URS1H位点，但它对单链URS1H的亲和力比对双链URS1H高得多，并且单杂交分析表明RPA在体内未检测到的水平下不结合该位点。此外，发现RPA中的条件致死突变对URS1H介导的抑制没有影响。这些结果表明，尽管RPA在体外结合URS1H，但它在体内似乎对通过该位点的转录抑制没有功能作用。
