Merkel T J, Nelson D M, Brauer C L, Kadner R J
Department of Microbiology, School of Medicine, University of Virginia, Charlottesville 22908.
J Bacteriol. 1992 May;174(9):2763-70. doi: 10.1128/jb.174.9.2763-2770.1992.
The uhpABCT locus of Escherichia coli encodes the transport system which allows the cell to accumulate a variety of sugar phosphates in unaltered form. The expression of uhpT, the gene encoding the transport protein, is regulated by the uhpABC gene products. The UhpA protein is required for expression; its deduced amino acid sequence shows that it belongs to a subfamily of bacterial transcription regulators including NarL, DegU, and FixJ. Members of this subfamily have an amino-terminal phosphorylation domain characteristic of so-called two-component regulators, such as OmpR, CheY, PhoB, and NtrC, and a carboxyl-terminal domain conserved among many transcriptional activators, including LuxR and MalT. The major sequence elements in the uhpT promoter that are needed for uhpT expression were investigated. Northern (RNA) hybridization analysis showed that the uhpT transcript was only present in cells induced for UhpT transport activity. The start site of transcription was identified by primer extension. Comparison of the regions upstream of the uhpT transcription start site in E. coli and Salmonella typhimurium suggested the presence of four sequence elements that might be involved in promoter function: a typical -10 region, a short inverted repeat centered at -32, a long inverted repeat centered at -64, and a cyclic AMP receptor protein-binding sequence centered at -103. Deletion and linker substitution mutations in the promoter demonstrated that the presence of the cyclic AMP receptor protein-binding site resulted in about an eightfold increase in promoter activity and that the -64, -32, and -10 elements were essential for promoter function. In vivo titration of transcriptional activator UhpA by the intact or mutant promoters on multicopy plasmids identified the -64 element as the UhpA-binding site. The two halves of the -64 inverted repeat did not contribute equally to promoter function and did not have to be intact for UhpA titration. The sequence recognized by UhpA is predicted to be 5' -GGCAAAACNNNGAAA.
大肠杆菌的uhpABCT基因座编码一种转运系统，该系统能使细胞以未改变的形式积累多种磷酸糖。编码转运蛋白的基因uhpT的表达受uhpABC基因产物的调控。UhpA蛋白是表达所必需的；其推导的氨基酸序列表明它属于细菌转录调节因子亚家族，包括NarL、DegU和FixJ。该亚家族成员具有氨基末端磷酸化结构域，这是所谓双组分调节因子（如OmpR、CheY、PhoB和NtrC）的特征，以及羧基末端结构域，该结构域在许多转录激活因子（包括LuxR和MalT）中保守。研究了uhpT启动子中uhpT表达所需的主要序列元件。Northern（RNA）杂交分析表明，uhpT转录本仅存在于诱导UhpT转运活性的细胞中。通过引物延伸确定了转录起始位点。比较大肠杆菌和鼠伤寒沙门氏菌中uhpT转录起始位点上游的区域，发现可能存在四个与启动子功能有关的序列元件：一个典型的-10区域、一个以-32为中心的短反向重复序列、一个以-64为中心的长反向重复序列和一个以-103为中心的环腺苷酸受体蛋白结合序列。启动子中的缺失和接头取代突变表明，环腺苷酸受体蛋白结合位点的存在使启动子活性增加约八倍，且-64、-32和-10元件对启动子功能至关重要。通过多拷贝质粒上完整或突变的启动子对转录激活因子UhpA进行体内滴定，确定-64元件为UhpA结合位点。-64反向重复序列的两半对启动子功能的贡献不均等，且UhpA滴定不一定需要其完整。预测UhpA识别的序列为5'-GGCAAAACNNNGAAA。
