Study on the transcriptional control of flagellar regulon in Salmonella typhimurium.

1.建立了沙门氏菌鞭毛调节子的整体结构。基于转录层次结构。鞭毛操纵子可分为三类(I类、II类和III类)。III类操纵子的转录受FliA基因产物的正调控，该基因产物属于II类，负调控由新发现的基因rflB的产物调控，该基因属于III.2类。对该基因进行了克隆和测序。推导的FliA蛋白的氨基酸序列与已知的RNA聚合酶的sigma因子有很大的同源性。将纯化的FliA蛋白加入含RNA聚合酶核心酶的体外转录系统，具有特异性地促进鞭毛操纵子启动子转录的活性。这表明FliA蛋白是鞭毛调节的另一种Sigma因子。对rflB基因进行了克隆和测序。将纯化的RflB蛋白加入含有FliA-RNA聚合酶的体外转录系统，具有特异性抑制鞭毛操纵子启动子转录的活性。分离到了一些III类操纵子的去抑制突变体。其中一些被发现存在fliA基因突变，这表明FliA和RflB蛋白之间可能存在分子相互作用。用纯化的FliA和RflB蛋白在体外进行了验证。在rflB突变体中，II类操纵子的表达增强。当将fliA缺陷引入相同的菌株中时，这种增强消失了。因此，整个鞭毛调节子的活性受FliA和RflB蛋白的正负调控。分别进行了分析。

1. An overall structure of flagellar regulon of Salmonella has been established. Based on the transcriptional hierarchy. flagellar operons can be divided into three classes (Class I. Class II, and Class III). The transcription of the class III operons is controlled positively by the gene product of fliA which belongs to class II and negatively by the product of a newly identified gene, rflB, which belongs to class III.2. The fliA gene was cloned and sequenced. The deduced animo acid sequence of the FliA protein was revealed to share a great deal of homology with those of the known sigma factors of RNA polymerase. The purified FliA protein had an activity to promote specifically the transcription from the promoter of flagellar operons when added to the in vitro transcription system containing RNA polymerase core enzyme. This indicates that the FliA protein is an alternative sigma factor specific for flagellar regulon.3. The rflB gene was cloned and sequenced. The purified RflB protein had an activity to repress specifically the transcription from the promoter of flagellar operons when added to the in vitro transcription system containing FliA-RNA polymerase.4. A number of derepressed mutants of the class III operons were isolated. Some of them were found to have mutations in the fliA gene, which suggests that there may be a molecular interaction between the FliA and the RflB proteins. This was proved in vitro using the purified FliA and RflB proteins.5. Expression of the class II operons was shown to be enhanced in the rflB mutants. This enhancement disappeared when the fliA defect was introduced into the same strains. Therefore, it has been concluded that the activity of the whole flagellar regulon is controlled positively and negatively by the FliA and the RflB proteins. respectively.