Flexible Recognition of the tRNA G18 Methylation Target Site by TrmH Methyltransferase through First Binding and Induced Fit Processes
Flexible Recognition of the tRNA G18 Methylation Target Site by TrmH Methyltransferase through First Binding and Induced Fit Processes
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DOI：
10.1074/jbc.m109.065698
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发表时间：
2010-03-19
影响因子：
4.8
通讯作者：
Hori, Hiroyuki
中科院分区：
文献类型：
--
作者：
Ochi, Anna;Makabe, Koki;Hori, Hiroyuki
转移RNA （Gm18）甲基转移酶（TrmH）催化甲基从s -腺苷- l-蛋氨酸转移到tRNA中保守的G18。我们研究了嗜热热菌TrmH对鸟苷靶标的识别机制。制备了13个酵母tRNAPhe突变体转录本，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、凝胶转移分析和抑制实验。甲基化位点通过RNA测序或引物延伸确定。虽然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严格识别鸟苷，当G18被dG或腺苷取代时，在相邻的G19上发生甲基化。TrmH甲基化d环上4到12个核苷酸长度的鸟苷，这表明鸟苷在d环内的位置选择相对灵活。我们的研究还表明，鸟嘌呤碱基的氧6原子是TrmH识别的积极决定因素。TrmH对底物的识别过程是诱导的，产物抑制的，含有Gm18的trna被TrmH排除在外。相反，用dG18取代G18会形成更稳定的TrmH-tRNA复合物。为了解决这一机制，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，即第一个双分子结合和随后的两个单分子诱导配合过程。
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 tRNAPhe 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.