Wu J, Benson A K, Newton A
Department of Molecular Biology, Princeton University, New Jersey 08544-1014, USA.
J Bacteriol. 1995 Jun;177(11):3241-50. doi: 10.1128/jb.177.11.3241-3250.1995.
Biosynthesis of the Caulobacter crescentus polar flagellum requires the expression of a large number of flagellar (fla) genes that are organized in a regulatory hierarchy of four classes (I to IV). The timing of fla gene expression in the cell cycle is determined by specialized forms of RNA polymerase and the appearance and/or activation of regulatory proteins. Here we report an investigation of the role of the C. crescentus transcriptional regulatory protein FlbD in the activation of sigma 54-dependent class III and class IV fla genes of the hierarchy by reconstituting transcription from these promoters in vitro. Our results demonstrate that transcription from promoters of the class III genes flbG, flgF, and flgI and the class IV gene fliK by Escherichia coli E sigma 54 is activated by FlbD or the mutant protein FlbDS140F (where S140F denotes an S-to-F mutation at position 140), which we show here has a higher potential for transcriptional activation. In vitro studies of the flbG promoter have shown previously that transcriptional activation by the FlbD protein requires ftr (ftr for flagellar transcription regulation) sequence elements. We have now identified multiple ftr sequences that are conserved in both sequence and spatial architecture in all known class III and class IV promoters. These newly identified ftr elements are positioned ca. 100 bp from the transcription start sites of each sigma 54-dependent fla gene promoter, and our studies indicate that they play an important role in controlling the levels of transcription from different class III and class IV promoters. We have also used mutational analysis to show that the ftr sequences are required for full activation by the FlbD protein both in vitro and in vivo. Thus, our results suggest that FlbD, which is encoded by the class II flbD gene, is a global regulator that activates the cell cycle-regulated transcription from all identified sigma 54-dependent promoters in the C. crescentus fla gene hierarchy.
新月柄杆菌极性鞭毛的生物合成需要大量鞭毛（fla）基因的表达，这些基因按照四个类别（I至IV）的调控层次进行组织。fla基因在细胞周期中的表达时间由RNA聚合酶的特殊形式以及调控蛋白的出现和/或激活决定。在此，我们报告了一项关于新月柄杆菌转录调控蛋白FlbD在激活该调控层次中依赖σ⁵⁴的III类和IV类fla基因方面作用的研究，通过在体外从这些启动子重建转录来进行研究。我们的结果表明，大肠杆菌E σ⁵⁴对III类基因flbG、flgF和flgI以及IV类基因fliK启动子的转录被FlbD或突变蛋白FlbDS140F（其中S140F表示第140位的丝氨酸突变为苯丙氨酸）激活，我们在此表明该突变蛋白具有更高的转录激活潜力。先前对flbG启动子的体外研究表明，FlbD蛋白的转录激活需要ftr（鞭毛转录调控的ftr）序列元件。我们现在已经鉴定出在所有已知的III类和IV类启动子的序列和空间结构中都保守的多个ftr序列。这些新鉴定的ftr元件位于每个依赖σ⁵⁴的fla基因启动子转录起始位点约100 bp处，我们的研究表明它们在控制不同III类和IV类启动子的转录水平中起重要作用。我们还通过突变分析表明，ftr序列在体外和体内都是FlbD蛋白完全激活所必需的。因此，我们的结果表明，由II类flbD基因编码的FlbD是一种全局调节因子，可激活新月柄杆菌fla基因调控层次中所有已鉴定的依赖σ⁵⁴的启动子的细胞周期调控转录。
