Conformational flexibility of viral RNA switches studied by FRET.
Conformational flexibility of viral RNA switches studied by FRET.
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DOI：
10.1016/j.ymeth.2015.09.013
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发表时间：
2015-12
期刊：
影响因子：
--
通讯作者：
Hermann T
中科院分区：
文献类型：
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作者：
Boerneke MA;Hermann T
参与转录和翻译调节的RNA开关的功能依赖于它们采用不同的、结构上明确定义的状态的能力。我们最近在正链RNA病毒中发现的一类新的配体响应RNA开关与传统的核糖开关不同。病毒开关响应于配体结合而经历大的构象变化，同时以其游离和配体结合形式保留相同的二级结构。在这里，我们描述FRET实验研究折叠和配体结合的病毒RNA开关。除了回顾以前的方法，涉及RNA模型构建体直接共轭与荧光染料，我们概述了新的模块化结构的设计和应用FRET实验，其中染料标记是通过杂交的核心RNA开关模块与通用DNA荧光探针。作为一个例子，折叠和配体结合的RNA开关从内部核糖体进入位点的丙型肝炎病毒进行了研究比较与传统的和模块化的FRET结构。
The function of RNA switches involved in the regulation of transcription and translation relies on their ability to adopt different, structurally well-defined states. A new class of ligand-responsive RNA switches, which we recently discovered in positive strand RNA viruses, are distinct from conventional riboswitches. The viral switches undergo large conformational changes in response to ligand binding while retaining the same secondary structure in their free and ligand-bound forms. Here, we describe FRET experiments to study folding and ligand binding of the viral RNA switches. In addition to reviewing previous approaches involving RNA model constructs which were directly conjugated with fluorescent dyes, we outline the design and application of new modular constructs for FRET experiments, in which dye labeling is achieved by hybridization of a core RNA switch module with universal DNA fluorescent probes. As an example, folding and ligand binding of the RNA switch from the internal ribosome entry site of hepatitis C virus is studied comparatively with conventional and modular FRET constructs.