Davis T L, Helinski D R, Roberts R C
Department of Biology, University of California, San Diego, La Jolla 92093-0634.
Mol Microbiol. 1992 Jul;6(14):1981-94. doi: 10.1111/j.1365-2958.1992.tb01371.x.
The broad-host-range plasmid RK2 has been shown to encode several proteins important for its maintenance within bacterial populations of a number of Gram-negative bacteria. Their genes are organized into two operons: parCBA and parD. These operons have been proposed to be transcribed from two divergent promoters, p-parCBA and p-parD, located within a sequence of approximately 150 bases. In this report we identify and characterize the sequences required for regulated transcription from these promoters in Escherichia coli, Agrobacterium tumefaciens and Pseudomonas aeruginosa. Both of these promoters are repressed by their own gene products in the same manner in all three bacteria tested, with ParA functioning as the primary repressor of p-parCBA and ParD functioning as the repressor of p-parD. The binding regions of these proteins were determined through deletion analyses, DNA mobility shift assays, and an examination of the effect of mutations in this region. Based on these observations, the ParA protein appears to bind to either two inverted repeat or two direct repeat sequences, one downstream from the transcriptional initiation site and the other upstream of the p-parCBA -35 box. The ParD protein appears to bind to one inverted repeat sequence, located between the -35 and -10 boxes of p-parD.
广宿主范围质粒RK2已被证明可编码几种对其在许多革兰氏阴性菌的细菌群体中维持自身存在很重要的蛋白质。它们的基因被组织成两个操纵子：parCBA和parD。有人提出这些操纵子是从位于大约150个碱基序列内的两个反向启动子p-parCBA和p-parD转录而来的。在本报告中，我们鉴定并表征了在大肠杆菌、根癌农杆菌和铜绿假单胞菌中从这些启动子进行调控转录所需的序列。在所有测试的三种细菌中，这两个启动子都以相同的方式被它们自己的基因产物抑制，ParA作为p-parCBA的主要阻遏物，ParD作为p-parD的阻遏物。通过缺失分析、DNA迁移率变动分析以及对该区域突变影响的检查确定了这些蛋白质的结合区域。基于这些观察结果，ParA蛋白似乎与两个反向重复序列或两个正向重复序列结合，一个在转录起始位点下游，另一个在p-parCBA -35框上游。ParD蛋白似乎与一个反向重复序列结合，该序列位于p-parD的-35和-10框之间。
