Pratt L A, Silhavy T J
Department of Molecular Biology, Princeton University, NJ 08544.
J Mol Biol. 1994 Nov 4;243(4):579-94. doi: 10.1016/0022-2836(94)90033-7.
In Escherichia coli, OmpR and EnvZ comprise a two component regulatory system that controls the relative expression of the outer membrane porin proteins, OmpF and OmpC. In this system, OmpR functions as a transcriptional regulator, serving as an activator of ompC, and as both an activator and a repressor of ompF. Previous evidence suggests that OmpR-mediated transcriptional activation involves direct interaction between OmpR and the C-terminal domain of the alpha subunit of RNA polymerase. However, it has remained unclear what region(s) of OmpR is directly involved in this proposed interaction. Moreover, little else is known about how OmpR activates transcription. To identify residues important for transcriptional activation, we screened for mutations in ompR that render the protein specifically defective in its ability to activate transcription. The isolated ompR alleles were characterized through haploid and diploid analyses at both the ompF and ompC promoters, and through an in vivo DNA binding assay. Through this approach, we have identified five amino acid residues in OmpR that are specifically required for transcriptional activation; R42, P179, E193, A196 and E198. We propose that these mutations define a region(s) in OmpR that may contact the C-terminal domain of alpha to mediate transcriptional activation.
在大肠杆菌中，OmpR和EnvZ构成一个双组分调节系统，该系统控制外膜孔蛋白OmpF和OmpC的相对表达。在这个系统中，OmpR作为转录调节因子发挥作用，充当ompC的激活剂以及ompF的激活剂和抑制剂。先前的证据表明，OmpR介导的转录激活涉及OmpR与RNA聚合酶α亚基的C末端结构域之间的直接相互作用。然而，OmpR的哪些区域直接参与这种假定的相互作用仍不清楚。此外，关于OmpR如何激活转录也知之甚少。为了鉴定对转录激活重要的残基，我们筛选了ompR中的突变，这些突变使该蛋白在激活转录的能力上出现特异性缺陷。通过在ompF和ompC启动子处进行单倍体和二倍体分析以及体内DNA结合试验，对分离出的ompR等位基因进行了表征。通过这种方法，我们在OmpR中鉴定出五个转录激活特别需要的氨基酸残基；R42、P179、E193、A196和E198。我们提出，这些突变定义了OmpR中可能与α的C末端结构域接触以介导转录激活的一个或多个区域。
