Transfer RNAs undergo diverse posttranscriptional modifications to regulate a myriad of cellular events including translation, stress response, and viral replication. These posttranscriptional modifications are synthesized by site-specific modification enzymes. Recent RNA-seq techniques have revealed multiple features of tRNA such as tRNA abundance, tRNA modification, and tRNA structure. Here, we adapt a tRNA-sequencing technique and design a new functional analysis where we perform mutational profiling of tRNA modifications to gain mechanistic insights into how tRNA modification enzymes recognize substrate tRNA. Profiling of Geobacillus stearothermophilus tRNAs and protein orthology analysis predict the existence of natural modifications in 44 tRNA molecular species of G. stearothermophilus. We selected the 1-methyladenosine modification at position 22 (m¹A22) and tRNA (m¹A22) methyltransferase (TrmK) for further analysis. Relative quantification of m¹A22 levels in 59 tRNA transcripts by mutational profiling reveals that TrmK selectively methylates a subset of tRNAs. Using 240 variants of tRNA^Leu transcripts, we demonstrate the conserved nucleosides including U8, A14, G15, G18, G19, U55, Purine57, and A58 are important for the methyl transfer reaction of TrmK. Additional biochemical experiments reveal that TrmK strictly recognizes U8, A14, G18, and U55 in tRNA. Furthermore, these findings from tRNA^Leu variants were crossvalidated using variants of three different tRNA species. Finally, a model of the TrmK–tRNA complex structure was constructed based on our findings and previous biochemical and structural studies by others. Collectively, our study expands functional analyses of tRNA modification enzyme in a high-throughput manner where our assay rapidly identifies substrates from a large pool of tRNAs.

转移 RNA 经历多种转录后修饰，以调节多种细胞事件，包括翻译、应激反应和病毒复制。这些转录后修饰是由位点特异性修饰酶合成的。最近的 RNA-seq 技术揭示了 tRNA 的多个特征，例如 tRNA 丰度、tRNA 修饰和 tRNA 结构。在这里，我们采用了 tRNA 测序技术并设计了一种新的功能分析，对 tRNA 修饰进行突变分析，以获得 tRNA 修饰酶如何识别底物 tRNA 的机制见解。嗜热脂肪地芽孢杆菌tRNA 分析和蛋白质直系同源分析预测嗜热脂肪地芽孢杆菌44 种 tRNA 分子物种中存在天然修饰。我们选择 22 位 1-甲基腺苷修饰 (m ¹ A22) 和 tRNA (m ¹ A22) 甲基转移酶 (TrmK) 进行进一步分析。通过突变分析对 59 个 tRNA 转录本中的 m ¹ A22 水平进行相对定量表明，TrmK 选择性甲基化 tRNA 的子集。使用 tRNA ^Leu转录本的 240 个变体，我们证明了保守核苷（包括 U8、A14、G15、G18、G19、U55、Purine57 和 A58）对于 TrmK 的甲基转移反应非常重要。其他生化实验表明，TrmK 严格识别 tRNA 中的 U8、A14、G18 和 U55。此外，tRNA ^Leu变体的这些发现使用三种不同 tRNA 物种的变体进行了交叉验证。最后，根据我们的发现以及其他人之前的生化和结构研究，构建了 TrmK-tRNA 复合结构的模型。 总的来说，我们的研究以高通量方式扩展了 tRNA 修饰酶的功能分析，其中我们的检测可以快速识别大量 tRNA 中的底物。