Department of Materials Science and Biotechnology, Graduate School of Science and Engineering, Ehime University, Bunkyo 3, Matsuyama, Ehime 790-8577..
J Biol Chem. 2010 Mar 19;285(12):9018-29. doi: 10.1074/jbc.M109.065698. Epub 2010 Jan 6.
Transfer RNA (Gm18) methyltransferase (TrmH) catalyzes methyl transfer from S-adenosyl-l-methionine to a conserved G18 in tRNA. We investigated the recognition mechanism of Thermus thermophilus TrmH for its guanosine target. Thirteen yeast tRNA(Phe) mutant transcripts were prepared in which the modification site and/or other nucleotides in the D-loop were substituted by dG, inosine, or other nucleotides. We then conducted methyl transfer kinetic studies, gel shift assays, and inhibition experiments using these tRNA variants. Sites of methylation were confirmed with RNA sequencing or primer extension. Although the G18G19 sequence is not essential for methylation by TrmH, disruption of G18G19 severely reduces the efficiency of methyl transfer. There is strict recognition of guanosine by TrmH, in that methylation occurs at the adjacent G19 when the G18 is replaced by dG or adenosine. The fact that TrmH methylates guanosine in D-loops from 4 to 12 nucleotides in length suggests that selection of the position of guanosine within the D-loop is relatively flexible. Our studies also demonstrate that the oxygen 6 atom of the guanine base is a positive determinant for TrmH recognition. The recognition process of TrmH for substrate is inducible and product-inhibited, in that tRNAs containing Gm18 are excluded by TrmH. In contrast, substitution of G18 with dG18 results in the formation of a more stable TrmH-tRNA complex. To address the mechanism, we performed the stopped-flow pre-steady state kinetic analysis. The result clearly showed that the binding of TrmH to tRNA is composed of at least three steps, the first bi-molecular binding and the subsequent two uni-molecular induced-fit processes.
转移 RNA (Gm18) 甲基转移酶 (TrmH) 催化 S-腺苷甲硫氨酸向 tRNA 中保守的 G18 转移甲基。我们研究了嗜热栖热菌 TrmH 对其鸟苷靶标的识别机制。我们制备了 13 种酵母 tRNA(Phe)突变转录本，其中 D 环中的修饰位点和/或其他核苷酸被 dG、肌苷或其他核苷酸取代。然后，我们使用这些 tRNA 变体进行了甲基转移动力学研究、凝胶迁移分析和抑制实验。使用 RNA 测序或引物延伸确定了甲基化位点。尽管 G18G19 序列不是 TrmH 甲基化所必需的，但 G18G19 的破坏严重降低了甲基转移的效率。TrmH 对鸟苷具有严格的识别，当 G18 被 dG 或腺苷取代时，甲基化发生在相邻的 G19 上。TrmH 甲基化 D 环中 4 到 12 个核苷酸长度的鸟苷表明，D 环中鸟苷位置的选择相对灵活。我们的研究还表明，鸟嘌呤碱基的氧 6 原子是 TrmH 识别的正决定因素。TrmH 对底物的识别过程是诱导和产物抑制的，即含有 Gm18 的 tRNA 被 TrmH 排斥。相比之下，用 dG18 取代 G18 会导致更稳定的 TrmH-tRNA 复合物的形成。为了解决这个问题，我们进行了停流预稳态动力学分析。结果清楚地表明，TrmH 与 tRNA 的结合至少由三个步骤组成，第一个是双分子结合，随后是两个单分子诱导契合过程。
