Abstract：
Transfer RNA (tRNA) delivers amino acids to the ribosome and functions as an essential adapter molecule for decoding codons on the messenger RNA (mRNA) during protein synthesis. Before attaining their proper activity, tRNAs undergo multiple post-transcriptional modifications with highly diversified roles such as stabilization of the tRNA structure, recognition of aminoacyl tRNA synthetases, precise codon-anticodon recognition, support of viral replication and onset of immune responses. The synthesis of the majority of modified nucleosides is catalyzed by a site-specific tRNA modification enzyme. This chapter provides a detailed protocol for using mutational profiling to analyze the enzymatic function of a tRNA methyltransferase in a high-throughput manner. In a previous study, we took tRNA m1A22 methyltransferase TrmK from Geobacillus stearothermophilus as a model tRNA methyltransferase and applied this protocol to gain mechanistic insights into how TrmK recognizes the substrate tRNAs. In theory, this protocol can be used unaltered for studying enzymes that catalyze modifications at the Watson-Crick face such as 1-methyladenosine (m1A), 3-methylcytosine (m3C), 3-methyluridine (m3U), 1-methylguanosine (m1G), and N2,N2-dimethylguanosine (m22G).
摘要：
转移RNA（tRNA）将氨基酸递送到核糖体，并在蛋白质合成过程中充当必需的衔接子分子，用于解码信使RNA（mRNA）上的密码子。在获得他们适当的活动之前，tRNA经历多种转录后修饰，具有高度多样化的作用，例如tRNA结构的稳定，氨基酰基tRNA合成酶的识别，精确的密码子-反密码子识别，支持病毒复制和免疫反应的开始。大多数修饰核苷的合成由位点特异性tRNA修饰酶催化。本章提供了使用突变谱分析以高通量方式分析tRNA甲基转移酶的酶促功能的详细方案。在之前的研究中,我们将来自嗜热脂肪土芽孢杆菌的tRNAm1A22甲基转移酶TrmK作为模型tRNA甲基转移酶,并应用该协议获得了有关TrmK如何识别底物tRNA的机制见解.理论上,该方案可用于研究在Watson-Crick脸上催化修饰的酶，如1-甲基腺苷（m1A），3-甲基胞嘧啶（m3C），3-甲基尿苷(m3U),1-甲基鸟苷(m1G),和N2,N2-二甲基鸟苷(m22G)。
