Laboratoire de Chimie Bactérienne, Institut de Microbiologie de la Méditerranée, Centre National de la Recherche Scientifique-Aix Marseille Université, Unité Mixte de Recherche, Marseille, France.
PLoS One. 2013 May 9;8(5):e63647. doi: 10.1371/journal.pone.0063647. Print 2013.
Controlling iron homeostasis is crucial for all aerobically grown living cells that are exposed to oxidative damage by reactive oxygen species (ROS), as free iron increases the production of ROS. Methionine sulfoxide reductases (Msr) are key enzymes in repairing ROS-mediated damage to proteins, as they reduce oxidized methionine (MetSO) residues to methionine. E. coli synthesizes two Msr, A and B, which exhibit substrate diastereospecificity. The bacterial iron-responsive small RNA (sRNA) RyhB controls iron metabolism by modulating intracellular iron usage. We show in this paper that RyhB is a direct regulator of the msrB gene that encodes the MsrB enzyme. RyhB down-regulates msrB transcripts along with Hfq and RNaseE proteins since mutations in the ryhB, fur, hfq, or RNaseE-encoded genes resulted in iron-insensitive expression of msrB. Our results show that RyhB binds to two sequences within the short 5'UTR of msrB mRNA as identified by reverse transcriptase and RNase and lead (II) protection assays. Toeprinting analysis shows that RyhB pairing to msrB mRNA prevents efficient ribosome binding and thereby inhibits translation initiation. In vivo site directed-mutagenesis experiments in the msrB 5'UTR region indicate that both RyhB-pairing sites are required to decrease msrB expression. Thus, this study suggests a novel mechanism of translational regulation where a same sRNA can basepair to two different locations within the same mRNA species. In contrast, expression of msrA is not influenced by changes in iron levels.
控制铁稳态对于所有需暴露于活性氧（ROS）引起的氧化损伤下的需氧生长的活细胞至关重要，因为游离铁会增加 ROS 的产生。甲硫氨酸亚砜还原酶（Msr）是修复 ROS 介导的蛋白质损伤的关键酶，因为它们将氧化的甲硫氨酸（MetSO）残基还原为甲硫氨酸。大肠杆菌合成两种 Msr，A 和 B，它们表现出底物对映体特异性。细菌铁反应性小 RNA（sRNA）RyhB 通过调节细胞内铁的利用来控制铁代谢。本文显示，RyhB 是编码 MsrB 酶的 msrB 基因的直接调节因子。由于 ryhB、fur、hfq 或 RNaseE 编码基因的突变导致 msrB 基因的铁不敏感表达，因此 RyhB 下调 msrB 转录本以及 Hfq 和 RNaseE 蛋白。我们的结果表明，RyhB 结合到 msrB mRNA 短 5'UTR 内的两个序列中，如通过逆转录酶和核糖核酸酶和铅（II）保护测定所鉴定的。足迹分析表明，RyhB 与 msrB mRNA 的配对阻止了核糖体的有效结合，从而抑制了翻译起始。msrB 5'UTR 区域的体内定点诱变实验表明，RyhB 配对位点都需要降低 msrB 的表达。因此，这项研究提出了一种新的翻译调控机制，其中相同的 sRNA 可以在同一 mRNA 物种内的两个不同位置进行碱基配对。相比之下，msrA 的表达不受铁水平变化的影响。
