鼠疫菌Fur蛋白对waaA的转录调控研究

Transcriptional regulation of waaA by Fur in Yersinia pestis

目的从转录水平上研究鼠疫耶尔森菌Fur蛋白对waaA的转录调控机制。方法提取鼠疫菌野生株(WT)和fur突变株(Δfur)的总RNA,采用引物延伸实验研究waaA的转录起始位点,并根据产物的丰度判断Fur蛋白对waaA的调控关系。构建克隆有waaA上游启动子区的LacZ重组质粒,将重组质粒转入WT和Δfur中,通过测定β-半乳糖苷酶活性来比较两株重组菌中的waaA启动子活性,进一步验证Fur蛋白对waaA的调控关系。PCR扩增waaA启动子区DNA序列全长,并纯化鼠疫菌His-Fur蛋白,通过体外的凝胶阻滞实验,即电泳迁移率变动实验(EMSA)和DNaseⅠ足迹实验验证His-Fur蛋白对waaA启动子区是否具有直接的结合作用。结果与结论 DNaseⅠ足迹实验表明,Fur蛋白对waaA启动子区的结合位置为-204至-150之间的碱基(翻译起始位点为+1);引物延伸实验显示waaA有一个转录起始位点T(-26),且该位点的转录活性直接被Fur蛋白激活。

Objective To study the transcriptional regulation mechanism of waaA by Fur in Yersinia pestis. Methods Primer extension assay was employed to detect the promoter activity （ the amount of primer extension product） of waaA in the wide-type （WT） strain and in the fur null mutant （Afur）. The waaA promoter-proximal region was cloned into pRWS0 containing a promoterless lacZ gene. The recombinant LacZ reporter plasmid was transformed into WT and Afur, respec- tively, to measure the promoter activity （ the [3-galactosidase activity） of waaA in WT and Afur using the [3-galactosidase enzyme assay system. The entire promoter-proximal region of the waaA gene was amplified by PCR from Y. pestis strain 201. At the same time, the over-expressed His-Fur was purified under native conditions with nickel loaded HiTrap Chela- ring Sepharose columns. And then, the electrophoretic mobihty shift assay （EMSA） and DNase I footprinting experiment were carried out to analyze the DNA-binding activity of His-Fur to waaA promoter region in vitro. Results and Conclusion The DNase I footprinting experiment showed that His-Fur binds to a single region from 204 bp to 150 bp upstream of waaA. Primer extension assay detected only one transcriptional start site located at 26 bp upstream of waaA, whose transcript is di- rectly activated by Fur.