Will William R, Frost Laura S
CW405 Biological Sciences Building, Department of Biological Sciences, University of Alberta, Edmonton, Alberta T6G 2E9, Canada.
J Bacteriol. 2006 Jan;188(1):124-31. doi: 10.1128/JB.188.1.124-131.2006.
The F plasmid of Escherichia coli allows horizontal DNA transfer between an F(+) donor cell and an F(-) recipient. Expression of the transfer genes is tightly controlled by a number of factors, including the following plasmid-encoded regulatory proteins: TraJ, the primary activator of the 33-kb tra operon, and the autoregulators TraM and TraY. Here, we demonstrate that the host RNA binding protein, Hfq, represses TraJ and TraM synthesis by destabilizing their respective mRNAs. Mating assays and immunoblot analyses for TraM and TraJ showed that transfer efficiency and protein levels increased in host cells containing a disruption in hfq compared to wild-type cells in stationary phase. The stability of transcripts containing a putative Hfq binding site located in the intergenic untranslated region between traM and traJ was increased in hfq mutant donor cells, suggesting that Hfq destabilizes these transcripts. Electrophoretic mobility shift assays demonstrated that Hfq specifically binds this region but not the antisense RNA, FinP, encoded on the opposite strand. Together, these findings indicate that Hfq regulates traM and traJ transcript stability by a mechanism separate from FinOP-mediated repression.
大肠杆菌的F质粒允许在F(+)供体细胞和F(-)受体细胞之间进行水平DNA转移。转移基因的表达受到多种因素的严格控制，包括以下质粒编码的调节蛋白：TraJ，33 kb tra操纵子的主要激活因子，以及自调节因子TraM和TraY。在这里，我们证明宿主RNA结合蛋白Hfq通过使TraJ和TraM各自的mRNA不稳定来抑制它们的合成。对TraM和TraJ的接合试验和免疫印迹分析表明，与处于稳定期的野生型细胞相比，在hfq基因发生破坏的宿主细胞中，转移效率和蛋白质水平有所提高。在hfq突变供体细胞中，包含位于traM和traJ之间基因间非翻译区的假定Hfq结合位点的转录本稳定性增加，这表明Hfq使这些转录本不稳定。电泳迁移率变动分析表明，Hfq特异性结合该区域，但不结合相反链上编码的反义RNA FinP。这些发现共同表明，Hfq通过一种与FinOP介导的抑制作用不同的机制调节traM和traJ转录本的稳定性。
