Friedrich-Schiller-Universität Jena, Biologisch-Pharmazeutische Fakultät, AG Bakteriengenetik, Philosophenweg 12, Jena D-07743, Germany.
Microbiology (Reading). 2011 Apr;157(Pt 4):1000-1008. doi: 10.1099/mic.0.047209-0. Epub 2011 Jan 20.
CopR is a transcriptional repressor encoded by the broad-host-range streptococcal plasmid pIP501, which also replicates in Bacillus subtilis. It acts in concert with the antisense RNA, RNAIII, to control pIP501 replication. CopR represses transcription of the essential repR mRNA about 10- to 20-fold. In previous work, DNA binding and dimerization constants were determined and the motifs responsible localized. The C terminus of CopR was shown to be required for stability. Furthermore, SELEX of the copR operator revealed that in vivo evolution was for maximal binding affinity. Here, we elucidate the repression mechanism of CopR. Competition assays showed that CopR-operator complexes are 18-fold less stable than RNA polymerase (RNAP)-pII complexes. DNase I footprinting revealed that the binding sites for CopR and RNAP overlap. Gel-shift assays demonstrated that CopR and B. subtilis RNAP cannot bind simultaneously, but compete for binding at promoter pII. Due to its higher intracellular concentration CopR inhibits RNAP binding. Additionally, KMnO(4) footprinting experiments indicated that prevention of open complex formation at pII does not further contribute to the repression effect of CopR.
CopR 是一种由广谱链球菌质粒细胞 pIP501 编码的转录抑制因子，也能在枯草芽孢杆菌中复制。它与反义 RNA RNAIII 协同作用来控制 pIP501 的复制。CopR 对必需的 repR mRNA 的转录有大约 10-20 倍的抑制作用。在以前的工作中，测定了 DNA 结合和二聚化常数，并定位了负责的基序。CopR 的 C 末端对于稳定性是必需的。此外，CopR 操纵子的 SELEX 表明，体内进化是为了最大的结合亲和力。在这里，我们阐明了 CopR 的抑制机制。竞争实验表明，CopR-操纵子复合物的稳定性比 RNA 聚合酶（RNAP）-pII 复合物低 18 倍。DNase I 足迹实验表明，CopR 和 RNAP 的结合位点重叠。凝胶迁移分析表明，CopR 和枯草芽孢杆菌 RNAP 不能同时结合，但在启动子 pII 处竞争结合。由于其在细胞内的浓度较高，CopR 抑制了 RNAP 的结合。此外，KMnO4 足迹实验表明，在 pII 处阻止开放复合物的形成并不能进一步增强 CopR 的抑制作用。
