Benson A K, Wu J, Newton A
Department of Molecular Biology, Princeton University, New Jersey 08540.
Res Microbiol. 1994 Jun-Aug;145(5-6):420-30. doi: 10.1016/0923-2508(94)90090-6.
The flagellar (fla) genes in Caulobacter crescentus are organized into a regulatory hierarchy of four levels (I-IV) in which transcription of the class III and class IV genes late in the cell cycle from sigma 54-dependent promoters depends on expression of the class II genes above them. The periodicity of fla gene expression has been attributed to sequential activation and repression by specific transcription factors. We have been particularly interested in understanding the function and regulation of one such transcription factor, FlbD. FlbD belongs to the NtrC family of bacterial response regulators that catalyse the initiation of transcription by sigma 54 RNA polymerase (E sigma 54) and its function is required for transcription of the class III and IV fla genes. Here we show that purified FlbD binds to ftr elements that are required for transcription from the sigma 54-dependent class III flbG promoter (ftr1) and repression of transcription from the class II fliF promoter (ftr4). Dimethylsulphate footprinting assays demonstrated that FlbD makes base-specific contacts at highly conserved guanine nucleotides in each half site of the ftr sequences. In a reconstituted in vitro transcription system using E. coli E sigma 54, we found that FlbD was clearly capable of driving transcriptional initiation from the flbG promoter and that this activity relied on the ftr1 binding site. Several observations suggest that phosphorylation plays a role in the regulation of FlbD activity. First, we found that a mutant form of FlbD (FlbDS140F) corresponding to the substitution found in a constitutively active NtrC protein (NtrCS160F), displayed a greater potential for activating E sigma 54-dependent transcription that the wildtype protein. We also observed that high energy-phosphate-containing molecules stimulate transcription activation by the wild type FlbD. Together, these results suggest that FlbD is responsible for mediating fla gene transcription initiation by E sigma 54 and that covalent modification is likely to play a role in governing FlbD activity during the cell cycle.
新月柄杆菌中的鞭毛（fla）基因被组织成一个四级调控层次结构（I-IV），其中细胞周期后期III类和IV类基因从依赖σ54的启动子转录依赖于其上游II类基因的表达。fla基因表达的周期性归因于特定转录因子的顺序激活和抑制。我们一直特别关注理解其中一种转录因子FlbD的功能和调控。FlbD属于细菌应答调节因子的NtrC家族，可催化由σ54 RNA聚合酶（Eσ54）启动转录，其功能是III类和IV类fla基因转录所必需的。在这里，我们表明纯化的FlbD与ftr元件结合，这些元件是从依赖σ54的III类flbG启动子（ftr1）转录以及抑制II类fliF启动子（ftr4）转录所必需的。硫酸二甲酯足迹分析表明，FlbD在ftr序列每个半位点高度保守的鸟嘌呤核苷酸处形成碱基特异性接触。在使用大肠杆菌Eσ54的重组体外转录系统中，我们发现FlbD显然能够驱动flbG启动子的转录起始，并且这种活性依赖于ftr1结合位点。一些观察结果表明磷酸化在FlbD活性调控中起作用。首先，我们发现与组成型活性NtrC蛋白（NtrCS160F）中发现的取代相对应的FlbD突变形式（FlbDS140F），比野生型蛋白具有更大的激活Eσ54依赖转录的潜力。我们还观察到含高能磷酸的分子刺激野生型FlbD的转录激活。总之，这些结果表明FlbD负责介导Eσ54引发的fla基因转录起始，并且共价修饰可能在细胞周期中调控FlbD活性方面发挥作用。
