Lai Stella M, Gopalan Venkat
Department of Chemistry and Biochemistry and Center for RNA Biology, The Ohio State University, Columbus, OH, USA.
Methods Mol Biol. 2021;2167:147-169. doi: 10.1007/978-1-0716-0716-9_9.
Kink-turns are important RNA structural modules that facilitate long-range tertiary interactions and form binding sites for members of the L7Ae family of proteins. Present in a wide variety of functional RNAs, kink-turns play key organizational roles in many RNA-based cellular processes, including translation, modification, and tRNA biogenesis. It is important to determine the contribution of kink-turns to the overall architecture of resident RNAs, as these modules dictate ribonucleoprotein (RNP) assembly and function. This chapter describes a site-directed, hydroxyl radical-mediated footprinting strategy that utilizes L7Ae-tethered chemical nucleases to experimentally validate computationally identified kink-turns in any RNA and under a wide variety of conditions. The work plan described here uses the catalytic RNase P RNA as an example to provide a blueprint for using this footprinting method to map RNA-protein interactions in other RNP complexes.
扭结转角是重要的RNA结构模块，可促进远距离三级相互作用，并形成L7Ae蛋白家族成员的结合位点。扭结转角存在于多种功能性RNA中，在许多基于RNA的细胞过程中发挥关键的组织作用，包括翻译、修饰和tRNA生物合成。确定扭结转角对驻留RNA整体结构的贡献很重要，因为这些模块决定核糖核蛋白（RNP）的组装和功能。本章描述了一种定点、羟基自由基介导的足迹分析策略，该策略利用与L7Ae相连的化学核酸酶，在各种条件下通过实验验证计算确定的任何RNA中的扭结转角。此处描述的工作计划以催化性RNase P RNA为例，为使用这种足迹分析方法绘制其他RNP复合物中的RNA-蛋白质相互作用提供了蓝图。
