Laboratory of Molecular Genetics, Department of Biology MCA, University of Camerino, 62032 Camerino, MC, Italy.
Nucleic Acids Res. 2010 Jun;38(10):3362-75. doi: 10.1093/nar/gkq025. Epub 2010 Feb 3.
The virulence gene icsA of Shigella flexneri encodes an invasion protein crucial for host colonization by pathogenic bacteria. Within the intergenic region virA-icsA, we have discovered a new gene that encodes a non-translated antisense RNA (named RnaG), transcribed in cis on the complementary strand of icsA. In vitro transcription assays show that RnaG promotes premature termination of transcription of icsA mRNA. Transcriptional inhibition is also observed in vivo by monitoring the expression profile in Shigella by real-time polymerase chain reaction and when RnaG is provided in trans. Chemical and enzymatic probing of the leader region of icsA mRNA either free or bound to RnaG indicate that upon hetero-duplex formation an intrinsic terminator, leading to transcription block, is generated on the nascent icsA mRNA. Mutations in the hairpin structure of the proposed terminator impair the RnaG mediated-regulation of icsA transcription. This study represents the first evidence of transcriptional attenuation mechanism caused by a small RNA in Gram-negative bacteria. We also present data on the secondary structure of the antisense region of RnaG. In addition, alternatively silencing icsA and RnaG promoters, we find that transcription from the strong RnaG promoter reduces the activity of the weak convergent icsA promoter through the transcriptional interference regulation.
福氏志贺菌的毒力基因 icsA 编码一种侵袭蛋白，对于致病菌对宿主的定植至关重要。在 virA-icsA 基因间隔区，我们发现了一个新基因，它编码一种非翻译的反义 RNA（命名为 RnaG），在 icsA 的互补链上顺式转录。体外转录实验表明，RnaG 促进 icsA mRNA 的转录过早终止。通过实时聚合酶链反应监测志贺氏菌中的表达谱，以及当 RnaG 以转位方式提供时，在体内也观察到转录抑制。对游离或与 RnaG 结合的 icsA mRNA 前导区进行化学和酶探测表明，形成异源双链后，在新生的 icsA mRNA 上会产生导致转录受阻的内在终止子。终止子发夹结构的突变会损害 RnaG 介导的 icsA 转录调控。本研究首次证明了在革兰氏阴性菌中，小 RNA 可引起转录衰减机制。我们还提供了关于 RnaG 反义区域二级结构的数据。此外，我们通过选择性沉默 icsA 和 RnaG 启动子发现，来自强 RnaG 启动子的转录通过转录干扰调节降低了弱汇聚的 icsA 启动子的活性。
