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Chemical Probing for Examining the Structure of Modified RNAs and Ligand Binding to RNA
Methods ( IF 4.8 ) Pub Date : 2019-03-01 , DOI: 10.1016/j.ymeth.2018.10.015
Prabuddha Waduge , Yogo Sakakibara , Christine S. Chow

Among different RNA modifications, the helix 69 (H69) region of the bacterial ribosomal RNA (rRNA) contains three pseudouridines (Ψs). H69 is functionally important due to its location in the heart of the ribosome. Several structural and functional studies have shown the importance of Ψ modifications in influencing the H69 conformation as well as maintaining key interactions in the ribosome during protein synthesis. Therefore, a need exists to understand the influence of modified nucleosides on conformational dynamics of the ribosome under solution conditions that mimic the cellular environment. In this review on chemical probing, we provide detailed protocols for the use of dimethyl sulfate (DMS) to examine H69 conformational states and the influence of Ψ modifications under varying solution conditions in the context of both ribosomal subunits and full ribosomes. The use of DMS footprinting to study the binding of aminoglycosides to the H69 region of bacterial rRNA as a potential antibiotic target will also be discussed. As highlighted in this work, DMS probing and footprinting are versatile techniques that can be used to gain important insight into RNA local structure and RNA-ligand interactions, respectively.

中文翻译:

用于检查修饰 RNA 的结构和与 RNA 结合的配体的化学探测

在不同的 RNA 修饰中,细菌核糖体 RNA (rRNA) 的螺旋 69 (H69) 区域包含三个假尿苷 (Ψs)。H69 在功能上很重要,因为它位于核糖体的中心。几项结构和功能研究表明,Ψ 修饰在影响 H69 构象以及维持蛋白质合成过程中核糖体中的关键相互作用方面具有重要意义。因此,需要了解修饰的核苷在模拟细胞环境的溶液条件下对核糖体构象动力学的影响。在这篇关于化学探测的评论中,我们提供了使用硫酸二甲酯 (DMS) 来检查 H69 构象状态和在核糖体亚基和完整核糖体的背景下不同溶液条件下 Ψ 修饰的影响的详细协议。还将讨论使用 DMS 足迹研究氨基糖苷类与细菌 rRNA 的 H69 区域的结合作为潜在的抗生素目标。正如在这项工作中所强调的,DMS 探测和足迹是通用技术,可用于分别获得对 RNA 局部结构和 RNA-配体相互作用的重要洞察。
更新日期:2019-03-01
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