大肠杆菌小RNA与RNA分子伴侣协同作用的新模式

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细菌为应对外界环境的变化(紫外辐射,氧化应激,热休克,低温,高渗透压,磷脂糖毒性,铁离子浓度)会产生非编码的小RNA。这些小RNA可以调节mRNA的翻译,或影响mRNA的稳定性。这种对外界环境变化的响应对细菌的生存是至关重要的。

rpoS mRNA编码RNA聚合酶σS因子。σs是大肠杆菌应激响应的主调节者。DsrA是一种小RNA,它在低温下调节rpoS的有效翻译。DsrA调节rpoS的翻译,需要RNA分子伴侣Hfq参与。我们解出了Hfq与DsrA上一段RNA片段(这是一段Hfq与DsrA结合区域),AU6A(AUUUUUUA), 以及与ADP三元复合物的晶体结构,以及Hfq与ADP二元复合物的晶体结构。发现Hfq与DsrA结合区域结合,及与ADP分子结合需要2个Hfq分子分别采用两个不同的分子表面协同作用。晶体结构显示AU6A与Hfq的结合采取了不同于已知的RNA与Hfq的结合模式。核磁共振化学位移扰动实验,顺磁弛豫增强实验,说明在溶液中Hfq与DsrA的结合,以及与ADP分子结合也采取相同模式。荧光共振能量转移实验表明全长的DsrA与全长的Hfq的结合确实涉及两个Hfq分子。上述工作还发现了前人未知的与ADP结合的关键氨基酸残基。

摘要:

Hfq is a bacterial post-transcriptional regulator. It facilitates base-pairing between sRNA and target mRNA. Hfq mediates DsrA-dependent translational activation of rpoS mRNA at low temperatures. rpoS encodes the stationary-phase σ factor σ(S), which is the central regulator in general stress response. However, structural information on Hfq-DsrA interaction is not yet available. Although Hfq is reported to hydrolyze ATP, the ATP-binding site is still unknown. Here, we report a ternary crystal complex structure of Escherichia coli Hfq bound to a major Hfq recognition region on DsrA (AU(6)A) together with ADP, and a crystal complex structure of Hfq bound to ADP. AU(6)A binds to the proximal and distal sides of two Hfq hexamers. ADP binds to a purine-selective site on the distal side and contacts conserved arginine or glutamine residues on the proximal side of another hexamer. This binding mode is different from previously postulated. The cooperation of two different Hfq hexamers upon nucleic acid binding in solution is verified by fluorescence polarization and solution nuclear magnetic resonance (NMR) experiments using fragments of Hfq and DsrA. Fluorescence resonance energy transfer conducted with full-length Hfq and DsrA also supports cooperation of Hfq hexamers upon DsrA binding. The implications of Hfq hexamer cooperation have been discussed.

文章原文链接:

Wang, W., Wang, L., Zou, Y., Zhang, J.,Gong, Q., Wu, J. & Shi, Y.* (2011). Cooperation of Escherichia coli Hfq hexamers in DsrA binding. Genes & Dev. 25, 2106-2117.

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