Inouye S, Gomada M, Sangodkar U M, Nakazawa A, Nakazawa T
Department of Biochemistry, Yamaguchi University School of Medicine, Japan.
J Mol Biol. 1990 Nov 20;216(2):251-60. doi: 10.1016/S0022-2836(05)80317-1.
Transcription of the first operon coding for m-xylene-degrading enzymes on the TOL plasmid of Pseudomonas putida is activated by the xylR gene product in the presence of m-xylene. The operon has the consensus sequence of the ntr/nif promoters at -24 and -12 regions, and the transcription is dependent on an RNA polymerase containing a sigma factor NtrA (RpoN or sigma 54). Deletion derivatives of the upstream sequence of the operon promoter were made in vitro and connected with the xylE gene on a plasmid. Their promoter activities were analyzed in Escherichia coli by monitoring catechol 2,3-dioxygenase activity, the xylE gene product. A cis-acting DNA element was identified, which is required for activation of the operon promoter by XylR protein in the presence of the inducer. This regulatory sequence of about 40 base-pairs in length was located 150 base-pairs upstream from the transcription start site. Analysis of the mutants containing insertions between the upstream regulatory sequence and the promoter sequence demonstrated strong dependence of the activation upon helical periodicity of DNA. The regulatory sequence functioned in the inverse orientation or at a distance of more than 1 x 10(3) base-pairs upstream from the promoter though less efficient. These results indicated that this upstream regulatory sequence might be the binding site for XylR protein. DNA-loop formation through protein-protein interaction between XylR protein attached to the upstream sequence and the NtrA-containing RNA polymerase bound by the promoter sequence was suggested for activation of the operon transcription. A sequence similar to the regulatory sequence of the first operon of xylene metabolism was found in the upstream region of the xylS gene, which is also activated by XylR protein in the presence of m-xylene.
恶臭假单胞菌TOL质粒上编码间二甲苯降解酶的首个操纵子，在间二甲苯存在时，其转录由xylR基因产物激活。该操纵子在-24和-12区域具有ntr/nif启动子的共有序列，转录依赖于含有σ因子NtrA（RpoN或σ54）的RNA聚合酶。在体外构建了该操纵子启动子上游序列的缺失衍生物，并将其与质粒上的xylE基因连接。通过监测儿茶酚2,3-双加氧酶活性（xylE基因产物），在大肠杆菌中分析了它们的启动子活性。鉴定出一个顺式作用DNA元件，它是诱导剂存在时XylR蛋白激活操纵子启动子所必需的。这个长度约为40个碱基对的调控序列位于转录起始位点上游150个碱基对处。对上游调控序列和启动子序列之间含有插入片段的突变体分析表明，激活作用强烈依赖于DNA的螺旋周期性。该调控序列以反向或在启动子上游超过1×10³个碱基对的距离起作用，尽管效率较低。这些结果表明，这个上游调控序列可能是XylR蛋白的结合位点。有人提出，通过附着在上游序列上的XylR蛋白与结合在启动子序列上的含NtrA的RNA聚合酶之间的蛋白质-蛋白质相互作用形成DNA环，从而激活操纵子转录。在xylS基因的上游区域发现了一个与二甲苯代谢首个操纵子调控序列相似的序列，该基因在间二甲苯存在时也由XylR蛋白激活。
