Expression génétique microbienne, UMR 8261, CNRS, Université de Paris, Institut de biologie physico-chimique (IBPC), Paris, France.
Methods Mol Biol. 2021;2298:307-323. doi: 10.1007/978-1-0716-1374-0_19.
During their biosynthesis, transfer RNAs (tRNAs) are decorated with a large number of posttranscriptional chemical modifications. Methods to directly detect the introduction of posttranscriptional modifications during tRNA maturation are rare and do not provide information on the temporality of modification events. Here, we report a methodology, using NMR as a tool to monitor tRNA maturation in a nondisruptive and continuous fashion in cellular extracts. This method requires the production of substrate tRNA transcripts devoid of modifications and active cell extracts containing the necessary cellular enzymatic activities to modify RNA. The present protocol describes these different aspects of our method and reports the time-resolved NMR monitoring of the yeast tRNA maturation as an example. The NMR-based methodology presented here could be adapted to investigate diverse features in tRNA maturation.
在生物合成过程中，转移 RNA（tRNA）会被大量的转录后化学修饰所修饰。直接检测 tRNA 成熟过程中引入转录后修饰的方法很少，并且无法提供修饰事件的时间性信息。在这里，我们报告了一种使用 NMR 作为工具，以非破坏和连续的方式在细胞提取物中监测 tRNA 成熟的方法。该方法需要产生无修饰的底物 tRNA 转录本和含有修饰 RNA 所需的必要细胞酶活性的活性细胞提取物。本方案描述了我们方法的这些不同方面，并报告了酵母 tRNA 成熟的时间分辨 NMR 监测作为示例。这里提出的基于 NMR 的方法可以进行修改，以研究 tRNA 成熟的不同特征。
