Dingwall A, Gober J W, Shapiro L
Department of Developmental Biology, Beckman Center, Stanford University School of Medicine, California 94305-5427.
J Bacteriol. 1990 Oct;172(10):6066-76. doi: 10.1128/jb.172.10.6066-6076.1990.
The genes that encode the components and regulatory proteins of the Caulobacter crescentus flagellum are transcribed at specific times in the cell cycle. One of these genes, flbN, is required early in the flagellar assembly process. The flbN gene was cloned and sequenced, and the time of transcription activation was determined. The derived amino acid sequence indicates that fibN encodes a 25-kilodalton protein with a cleavable leader peptide. The flbN-encoded protein has 30.8% identity with the protein encoded by the Salmonella typhimurium basal body L-ring gene, flgH. Site-directed mutagenesis and gel mobility shift assays identified a binding site at -100 from the transcription start site for a trans-acting protein, RF-2, that functions to partially activate flbN transcription at a defined time in the cell cycle. The RF-2 binding region is similar to a NifA binding site normally used in the activation of some sigma 54 promoters involved in nitrogen fixation in other bacteria. Transcription of a flbN-reporter gene fusion in an Escherichia coli background was dependent on the presence of a NifA transcription factor supplied by a plasmid-borne Rhizobium meliloti gene encoding NifA. A deletion or base changes in the RF-2 binding region eliminated expression of the flbN gene in E. coli even when a NifA protein was provided in trans, suggesting that a sigma 54 promoter with an upstream activator element is used by the C. crescentus flbN gene. A consensus sequence for a sigma 54 promoter was found at the appropriate distance 5' to one of two identified transcription start sites. Site-directed mutagenesis confirmed that a conserved nucleotide in this sigma 54 promoter consensus sequence was required for transcription. Deletion of the region 5' to the apparent sigma 54 promoter caused a complete loss of transcription activation. Transcription activation of flbN in C. crescentus involves the combination of several elements: the NifA-like site is required for full activation, and other sequence elements 5' to the promoter and 3' to the transcription start site are necessary for the correct time of transcription initiation.
编码新月柄杆菌鞭毛组件和调控蛋白的基因在细胞周期的特定时间转录。其中一个基因flbN在鞭毛组装过程的早期是必需的。克隆并测序了flbN基因，并确定了转录激活时间。推导的氨基酸序列表明，fibN编码一种带有可裂解前导肽的25千道尔顿蛋白。flbN编码的蛋白与鼠伤寒沙门氏菌基体L环基因flgH编码的蛋白有30.8%的同一性。定点诱变和凝胶迁移率变动分析确定了一个反式作用蛋白RF-2在转录起始位点上游-100处的结合位点，该蛋白在细胞周期的特定时间部分激活flbN转录。RF-2结合区域类似于通常用于激活其他细菌中参与固氮的一些σ54启动子的NifA结合位点。在大肠杆菌背景中flbN报告基因融合体的转录依赖于由质粒携带的编码NifA的苜蓿根瘤菌基因提供的NifA转录因子的存在。RF-2结合区域的缺失或碱基变化消除了大肠杆菌中flbN基因的表达，即使反式提供NifA蛋白也是如此，这表明新月柄杆菌flbN基因使用了带有上游激活元件的σ54启动子。在两个已确定的转录起始位点之一的5'端适当距离处发现了σ54启动子的共有序列。定点诱变证实该σ54启动子共有序列中的一个保守核苷酸是转录所必需的。明显的σ54启动子5'端区域的缺失导致转录激活完全丧失。新月柄杆菌中flbN的转录激活涉及几个元件的组合：NifA样位点是完全激活所必需的，启动子5'端和转录起始位点3'端的其他序列元件对于正确的转录起始时间是必需的。
