A ribosome profiling study of mRNA cleavage by the endonuclease RelE.
A ribosome profiling study of mRNA cleavage by the endonuclease RelE.
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DOI：
10.1093/nar/gkw944
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发表时间：
2017-01-09
影响因子：
14.9
通讯作者：
Buskirk AR
中科院分区：
文献类型：
--
作者：
Hwang JY;Buskirk AR
RelE与细菌中的持久性和应激反应途径有关，它通过切割核糖体A位点内的mRNA来关闭蛋白质合成。结构和生物化学研究表明，RelE切割具有一定的序列特异性，我们在这里进一步表征，并且它在核糖体的背景之外没有显示出活性。我们通过核糖体分析获得了RelE对翻译的影响的全局视图，观察到核糖体通过mRNA切割、核糖体拯救和起始的动态循环在基因的5′端积累。此外，将纯化的RelE添加到细胞裂解物中显示出作为用于产生核糖体足迹的方法的前景。在细菌中，由于MNase消化（用于降解mRNA未受保护区域的方法）固有的问题，谱分析研究的分辨率相对较低，并且没有产生关于阅读框架的信息。相反，我们发现RelE产生精确的3′-末端，这是第一次揭示细菌中的阅读框架。鉴于RelE已被证明在生命的所有三个领域中发挥作用，RelE具有改善阅读框架的潜力，并在核糖体分析实验中更广泛地阐明A位点占据。
Implicated in persistence and stress response pathways in bacteria, RelE shuts down protein synthesis by cleaving mRNA within the ribosomal A site. Structural and biochemical studies have shown that RelE cuts with some sequence specificity, which we further characterize here, and that it shows no activity outside the context of the ribosome. We obtained a global view of the effect of RelE on translation by ribosome profiling, observing that ribosomes accumulate on the 5′-end of genes through dynamic cycles of mRNA cleavage, ribosome rescue and initiation. Moreover, the addition of purified RelE to cell lysates shows promise as a method for generating ribosome footprints. In bacteria, profiling studies have suffered from relatively low resolution and have yielded no information on reading frame due to problems inherent to MNase digestion, the method used to degrade unprotected regions of mRNA. In contrast, we find that RelE yields precise 3′-ends that for the first time reveal reading frame in bacteria. Given that RelE has been shown to function in all three domains of life, RelE has potential to improve reading frame and shed light on A-site occupancy in ribosome profiling experiments more broadly.