Riboswitch structure and dynamics by smFRET microscopy.
Riboswitch structure and dynamics by smFRET microscopy.
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DOI：
10.1016/b978-0-12-801122-5.00015-5
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发表时间：
2014
影响因子：
--
通讯作者：
Walter NG
中科院分区：
文献类型：
--
作者：
Suddala KC;Walter NG
核糖开关是一种结构化的非编码RNA元件，通过感知不同代谢物的细胞内浓度来控制其嵌入信使RNA的表达。顾名思义，核糖开关本质上是动态的，因此研究它们固有的构象动力学和配体介导的折叠对于理解它们的作用机制是重要的。单分子荧光能量转移显微镜(SmFRET)是研究生物大分子，特别是RNA的折叠途径、分子内和分子间动力学的一种强大而通用的技术。SmFRET监测分子内距离及其时间演化的能力使其成为探测核糖开关结构和动力学的特别有洞察力的工具。在这里，我们详细介绍了使用基于棱镜的全内反射荧光(TIRF)显微镜研究核糖开关的结构、动力学和配体结合机制的一般步骤。
Riboswitches are structured non-coding RNA elements that control the expression of their embedding messenger RNAs by sensing the intracellular concentration of diverse metabolites. As the name suggests, riboswitches are dynamic in nature so that studying their inherent conformational dynamics and ligand-mediated folding is important for understanding their mechanism of action. Single molecule fluorescence energy transfer (smFRET) microscopy is a powerful and versatile technique for studying the folding pathways and intra- and intermolecular dynamics of biological macromolecules, especially RNA. The ability of smFRET to monitor intramolecular distances and their temporal evolution make it a particularly insightful tool for probing the structure and dynamics of riboswitches. Here, we detail the general steps for using prism-based total internal reflection fluorescence (TIRF) microscopy for smFRET studies of the structure, dynamics and ligand binding mechanisms of riboswitches.