Kimata K, Inada T, Tagami H, Aiba H
Department of Molecular Biology, Graduate School of Science, Nagoya University, Chikusa, Japan.
Mol Microbiol. 1998 Sep;29(6):1509-19. doi: 10.1046/j.1365-2958.1998.01035.x.
Glucose stimulates the expression of ptsG encoding the major glucose transporter in Escherichia coli. We isolated Tn 10 insertion mutations that confer constitutive expression of ptsG. The mutated gene was identified as mlc, encoding a protein that is known to be a repressor for transcription of several genes involved in carbohydrate utilization. Expression of ptsG was eliminated in a mlc crp double-negative mutant. The Mlc protein was overproduced and purified. In vitro transcription studies demonstrated that transcription of ptsG is stimulated by CRP-cAMP and repressed by Mlc. The action of Mlc is dominant over that of CRP-cAMP. DNase I footprinting experiments revealed that CRP-cAMP binds at two sites centred at -40.5 and -95.5 and that Mlc binds at two regions centred around -8 and -175. The binding of CRP-cAMP stimulated the binding of RNA polymerase to the promoter while Mlc inhibited the binding of RNA polymerase but not the binding of CRP-cAMP. Gel-mobility shift assay indicated that glucose does not affect the Mlc binding to the ptsG promoter. Our results suggest that Mlc is responsible for the repression of ptsG transcription and that glucose modulates the Mlc activity by unknown mechanism.
葡萄糖可刺激大肠杆菌中编码主要葡萄糖转运蛋白的ptsG的表达。我们分离出了赋予ptsG组成型表达的Tn10插入突变。突变基因被鉴定为mlc，它编码一种已知为参与碳水化合物利用的几个基因转录的阻遏物的蛋白质。在mlc crp双阴性突变体中，ptsG的表达被消除。Mlc蛋白被过量表达并纯化。体外转录研究表明，ptsG的转录受CRP-cAMP刺激并受Mlc抑制。Mlc的作用比CRP-cAMP的作用更占优势。DNase I足迹实验表明，CRP-cAMP结合在以-40.5和-95.5为中心的两个位点，而Mlc结合在以-8和-175为中心的两个区域。CRP-cAMP的结合刺激了RNA聚合酶与启动子的结合，而Mlc抑制了RNA聚合酶的结合，但不抑制CRP-cAMP的结合。凝胶迁移率变动分析表明，葡萄糖不影响Mlc与ptsG启动子的结合。我们的结果表明，Mlc负责ptsG转录的抑制，并且葡萄糖通过未知机制调节Mlc活性。
