Cycling of the Sm-like protein Hfq on the DsrA small regulatory RNA
Cycling of the Sm-like protein Hfq on the DsrA small regulatory RNA
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DOI：
10.1016/j.jmb.2004.10.006
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发表时间：
2004-12-10
影响因子：
5.6
通讯作者：
Woodson, SA
中科院分区：
文献类型：
--
作者：
Lease, RA;Woodson, SA
小rna （sRNAs）调节细菌参与环境适应的基因。这种RNA调控需要Hfq，一种细菌sm样蛋白，稳定sRNAs并增强RNA-RNA相互作用。为了了解sRNAs识别靶标的机制，我们研究了Hfq、sRNA DsrA及其调控靶标rpoS mRNA（编码应激反应sigma因子）之间的相互作用。核酸酶足迹显示Hfq识别rpoS mRNA中的多个位点，而不会显著干扰抑制翻译起始的5'先导子的二级结构。然而，与DsrA的碱基配对使rpoS核糖体结合位点完全可接近，正如遗传数据预测的那样。在凝胶迁移实验中，Hfq结合DsrA的紧密程度是DsrA (.) rpoS RNA复合物的四倍。因此，当DsrA碱基与rpoS mRNA配对时，由于DsrA的构象变化，Hfq迅速从其在DsrA上的高亲和力结合位点移位。Hfq加速了DsrA (.) rpoS RNA结合，并使RNA复合物稳定了两倍。当Hfq占据游离DsrA的主要结合位点时，DsrA和rpoS mRNA的杂交效果最佳，而当Hfq与DsrA (.) rpoS RNA复合物结合时，DsrA和rpoS mRNA的杂交效果受到抑制。我们得出结论，Hfq结合到自由的DsrA sRNA上，然后从sRNA (.) mRNA复合物中释放Hfq，从而刺激rpoS mRNA的识别。(C) 2004 Elsevier Ltd.版权所有。
Small RNAs (sRNAs) regulate bacterial genes involved in environmental adaptation. This RNA regulation requires Hfq, a bacterial Sm-like protein that stabilizes sRNAs and enhances RNA-RNA interactions. To understand the mechanism of target recognition by sRNAs, we investigated the interactions between Hfq, the sRNA DsrA, and its regulatory target rpoS mRNA, which encodes the stress response sigma factor. Nuclease footprinting revealed that Hfq recognized multiple sites in rpoS mRNA without significantly perturbing secondary structure in the 5' leader that inhibits translation initiation. Base-pairing with DsrA, however, made the rpoS ribosome binding site fully accessible, as predicted by genetic data. Hfq bound DsrA four times more tightly than the DsrA (.) rpoS RNA complex in gel mobility-shift assays. Consequently, Hfq is displaced rapidly from its high-affinity binding site on DsrA by conformational changes in DsrA, when DsrA base-pairs with rpoS mRNA. Hfq accelerated DsrA (.) rpoS RNA association and stabilized the RNA complex up to twofold. Hybridization of DsrA and rpoS mRNA was optimal when Hfq occupied its primary binding site on free DsrA, but was inhibited when Hfq associated with the DsrA (.) rpoS RNA complex. We conclude that recognition of rpoS mRNA is stimulated by binding of Hfq to free DsrA sRNA, followed by release of Hfq from the sRNA (.) mRNA complex. (C) 2004 Elsevier Ltd. All rights reserved.