TrmH is a eubacterial tRNA methyltransferase responsible for formation of 2’-O-methylguaosine at position 18 (Gm18) in tRNA. In Escherichia coli cells, only 14 tRNA species possess the Gm18 modification. To investigate the substrate tRNA selection mechanism of E. coli TrmH, we performed biochemical and structural studies. E. coli TrmH requires a high concentration of substrate tRNA for efficient methylation. Experiments using native tRNASerCGA purified from a trmH gene disruptant strain showed that modified nucleosides do not affect the methylation. A gel mobility-shift assay reveals that TrmH captures tRNAs without distinguishing between relatively good and very poor substrates. Methylation assays using wild-type and mutant tRNA transcripts revealed that the location of G18 in the D-loop is very important for efficient methylation by E. coli TrmH. In the case of tRNASer, tRNATyr and tRNALeu, the D-loop structure formed by interaction with the long variable region is important. For tRNAGln, the short distance between G18 and A14 is important. Thus, our biochemical study explains all Gm18 modification patterns in E. coli tRNAs. The crystal structure of E. coli TrmH has also been solved, and the tRNA binding mode of E. coli TrmH is discussed based on the structure.

中文翻译：

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大肠杆菌 tRNA (Gm18) 甲基转移酶，TrmH 需要其甲基化位点 (G18) 在 D 环中正确定位才能有效甲基化

TrmH 是一种真细菌 tRNA 甲基转移酶，负责在 tRNA 的 18 位 (Gm18) 处形成 2'-O-甲基鸟苷。在大肠杆菌细胞中，只有 14 种 tRNA 具有 Gm18 修饰。为了研究大肠杆菌 TrmH 的底物 tRNA 选择机制，我们进行了生化和结构研究。大肠杆菌 TrmH 需要高浓度的底物 tRNA 才能有效甲基化。使用从 trmH 基因破坏菌株纯化的天然 tRNASerCGA 进行的实验表明，修饰的核苷不会影响甲基化。凝胶迁移率变化测定表明，TrmH 捕获 tRNA 时不区分相对较好和非常差的底物。使用野生型和突变型 tRNA 转录本进行的甲基化测定表明，G18 在 D 环中的位置对于大肠杆菌 TrmH 的有效甲基化非常重要。就 tRNASer、tRNATyr 和 tRNALeu 而言，与长可变区相互作用形成的 D 环结构很重要。对于 tRNAGln，G18 和 A14 之间的短距离很重要。因此，我们的生化研究解释了大肠杆菌 tRNA 中的所有 Gm18 修饰模式。大肠杆菌TrmH的晶体结构也已得到解答，并根据该结构讨论了大肠杆菌TrmH的tRNA结合模式。