Schneiders Thamarai, Levy Stuart B
Center for Adaptation Genetics and Drug Resistance and the Department of Molecular Biology and Microbiology, Tufts University School of Medicine, 136 Harrison Avenue, Boston, MA 02111, USA.
J Biol Chem. 2006 Apr 14;281(15):10049-55. doi: 10.1074/jbc.M512097200. Epub 2006 Feb 14.
The Escherichia coli transcriptional regulator MarA affects functions that include antibiotic resistance, persistence, and survival. MarA functions as an activator or repressor of transcription utilizing similar degenerate DNA sequences (marboxes) with three different binding site configurations with respect to the RNA polymerase-binding sites. We demonstrate that MarA down-regulates rob transcripts both in vivo and in vitro via a MarA-binding site within the rob promoter that is positioned between the -10 and -35 hexamers. As for the hdeA and purA promoters, which are repressed by MarA, the rob marbox is also in the "backward" orientation. Protein-DNA interactions show that SoxS and Rob, like MarA, bind the same marbox in the rob promoter. Electrophoretic mobility shift analyses with a MarA-specific antibody demonstrate that MarA and RNA polymerase form a ternary complex with the rob promoter DNA. Transcription experiments in vitro and potassium permanganate footprinting analysis show that MarA affects the RNA polymerase-mediated closed to open complex formation at the rob promoter.
大肠杆菌转录调节因子MarA影响包括抗生素抗性、持留性和生存能力等多种功能。MarA作为转录激活因子或抑制因子发挥作用，它利用与RNA聚合酶结合位点具有三种不同结合位点构型的类似简并DNA序列（marbox）。我们证明，MarA在体内和体外均可通过位于rob启动子中-10和-35六聚体之间的MarA结合位点下调rob转录本。与受MarA抑制的hdeA和purA启动子一样，rob marbox也呈“反向”取向。蛋白质-DNA相互作用表明，SoxS和Rob与MarA一样，结合rob启动子中的相同marbox。用MarA特异性抗体进行的电泳迁移率变动分析表明，MarA和RNA聚合酶与rob启动子DNA形成三元复合物。体外转录实验和高锰酸钾足迹分析表明，MarA影响RNA聚合酶介导的rob启动子处从封闭复合物到开放复合物的形成。
