Two targets in pCF10 DNA for PrgX binding:: Their role in production of Qa and prgX mRNA and in regulation of pheromone-inducible conjugation
Two targets in pCF10 DNA for PrgX binding:: Their role in production of Qa and prgX mRNA and in regulation of pheromone-inducible conjugation
复制标题
DOI：
10.1006/jmbi.2001.5294
复制
发表时间：
2002-02-01
影响因子：
5.6
通讯作者：
Dunny, GM
中科院分区：
文献类型：
--
作者：
Bae, T;Kozlowicz, B;Dunny, GM
PrgX是参与粪肠球菌质粒pCF10信息素诱导偶联功能负调控的主要细胞质蛋白。PrgX被认为与一种名为Qa的反义RNA协同作用，抑制prgQ启动子转录到介导偶联功能的pCF10基因的读通；PrgX也正调控自身的表达，以及Qa的表达。我们在pCF10的PrgX和prgQ基因之间的基因间区发现了两个PrgX结合的DNA靶点。prgX附近的主要结合位点包括一个11bp的回文序列，对His-tagged prgX （His-PrgX）具有较高的亲和力。次级结合位点位于prgQ启动子的-35和-10区域之间，只包含一半的回文序列；该结合位点的亲和力较弱。包含prgQ启动子和次级结合位点的pCF10区域大大降低了Qa RNA水平，这种降低被主要结合位点和PrgX的存在所克服。在结合位点单独或联合突变的构建体中，PrgX蛋白和Qa RNA的细胞内水平显著降低。基于这些结果，我们提出两个DNA结合位点都是PrgX表达的自动调节和Qa RNA的正调节所必需的。(C) 2002爱思唯尔科学有限公司
PrgX is the primary cytoplasmic protein involved in negative control of pheromone-inducible conjugation functions of the Enterococcus faecalis plasmid pCF10. PrgX is believed to act in concert with an antisense RNA called Qa to inhibit readthrough of transcription from the prgQ promoter into the pCF10 genes mediating conjugation functions; PrgX also positively regulates its own expression, as well as that of Qa. We found two DNA target sites for PrgX binding in the intergenic region between the prgX and prgQ genes of pCF10. The primary binding site near prgX includes an 11 bp palindromic sequence and showed relatively high affinity for His-tagged PrgX (His-PrgX). The secondary binding site is between the -35 and -10 regions of the prgQ promoter, and contains only a half of the palindromic sequence; this binding site showed weaker affinity. A region of pCF10 including the prgQ promoter and the secondary binding site reduced Qa RNA levels greatly and this reduction was overcome by the presence of the primary binding site and PrgX. In constructs where the binding sites were mutated individually or in combination, the intracellular levels of PrgX protein and Qa RNA were reduced significantly. On the basis of these results, we propose that both DNA binding sites are required for the autoregulation of PrgX expression and for positive regulation of Qa RNA. (C) 2002 Elsevier Science Limited.