Substrate tRNA Recognition Mechanism of Eubacterial tRNA (m1A58) Methyltransferase (TrmI)*
Substrate tRNA Recognition Mechanism of Eubacterial tRNA (m1A58) Methyltransferase (TrmI)*
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DOI：
10.1074/jbc.m114.606038
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发表时间：
2015-01
期刊：
影响因子：
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通讯作者：
Hiroyuki Takuma;Natsumi Ushio;Masayuki Minoji;Ai Kazayama;Naoki Shigi;A. Hirata;Chie Tomikawa;A. Ochi;H. Hori
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文献类型：
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作者：
Hiroyuki Takuma;Natsumi Ushio;Masayuki Minoji;Ai Kazayama;Naoki Shigi;A. Hirata;Chie Tomikawa;A. Ochi;H. Hori
背景：tRNA甲基转移酶特异性识别底物tRNAs。结果：为了阐明TrmI的tRNA识别机制，我们在体内外分析了3种tRNA和45种tRNA变异体。结论：TrmI可识别tRNA T环中的氨酰基、可变区、C56、嘌呤57、A58和U60。意义：我们的体外实验结果解释了体内tRNAs甲基化水平的调节。TrmI在tRNA的第58位(M1A58)产生N1-甲基腺苷。嗜热嗜热菌tRNAPhe转录本被嗜热性嗜热菌TrmI有效甲基化，而酵母tRNAPhe转录本甲基化较差。从这两个tRNAs获得的14个嵌合tRNA转录本表明，TrmI识别氨基酰茎、可变区和T-loop的组合。10个tRNA缺失变体证实了这一点：含有氨基酰茎、可变区和T-臂的TrmI甲基化转录本。通过破坏T-干，确认了对T-干本身的要求。破坏T-臂和D-臂之间的相互作用加速了甲基化，表明这种破坏包括在反应的一部分。对17个点突变转录本的实验阐明了正序列决定簇C56、嘌呤57、A58和U60。用肌苷和2-氨基嘌呤取代A58完全取消了甲基化，证明A58中的6-氨基被TrmI识别。T.thermophilus tRNAGGUThrGGUThr含有C60而不是U60。TRNAGGUThr转录本被TrmI甲基化程度很低，用U取代C60增加了甲基化，这与点突变实验一致。凝胶漂移实验表明，tRNAGGUThr对TrmI的亲和力低于tRNAPhe。此外，对从trmI基因中断菌株中纯化的tRNAGGUThr的分析表明，tRNA的其他修饰促进了TrmI形成m1A58。此外，野生型tRNAGGUThr的核苷分析表明，不到50%的tRNAGGUThr含有m1A58。因此，体外实验的结果得到了体内甲基化模式的证实。
Background: tRNA methyltransferases specifically recognize substrate tRNAs. Results: To clarify the tRNA recognition mechanism of TrmI, three tRNA species and 45 variants were analyzed in vitro and in vivo. Conclusion: TrmI recognizes the aminoacyl stem, variable region, C56, purine 57, A58, and U60 in the T-loop of tRNA. Significance: Our in vitro experimental results explain the regulation of in vivo methylation levels in tRNAs. TrmI generates N1-methyladenosine at position 58 (m1A58) in tRNA. The Thermus thermophilus tRNAPhe transcript was methylated efficiently by T. thermophilus TrmI, whereas the yeast tRNAPhe transcript was poorly methylated. Fourteen chimeric tRNA transcripts derived from these two tRNAs revealed that TrmI recognized the combination of aminoacyl stem, variable region, and T-loop. This was confirmed by 10 deletion tRNA variants: TrmI methylated transcripts containing the aminoacyl stem, variable region, and T-arm. The requirement for the T-stem itself was confirmed by disrupting the T-stem. Disrupting the interaction between T- and D-arms accelerated the methylation, suggesting that this disruption is included in part of the reaction. Experiments with 17 point mutant transcripts elucidated the positive sequence determinants C56, purine 57, A58, and U60. Replacing A58 with inosine and 2-aminopurine completely abrogated methylation, demonstrating that the 6-amino group in A58 is recognized by TrmI. T. thermophilus tRNAGGUThrGGUThr contains C60 instead of U60. The tRNAGGUThr transcript was poorly methylated by TrmI, and replacing C60 with U increased the methylation, consistent with the point mutation experiments. A gel shift assay revealed that tRNAGGUThr had a low affinity for TrmI than tRNAPhe. Furthermore, analysis of tRNAGGUThr purified from the trmI gene disruptant strain revealed that the other modifications in tRNA accelerated the formation of m1A58 by TrmI. Moreover, nucleoside analysis of tRNAGGUThr from the wild-type strain indicated that less than 50% of tRNAGGUThr contained m1A58. Thus, the results from the in vitro experiments were confirmed by the in vivo methylation patterns.