Aquifex aeolicus tRNA (N2, N2-Guanine)-dimethyltransferase (Trm1) Catalyzes Transfer of Methyl Groups Not Only to Guanine 26 but Also to Guanine 27 in tRNA
Aquifex aeolicus tRNA (N2, N2-Guanine)-dimethyltransferase (Trm1) Catalyzes Transfer of Methyl Groups Not Only to Guanine 26 but Also to Guanine 27 in tRNA
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DOI：
10.1074/jbc.m109.020024
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发表时间：
2009-07-31
影响因子：
4.8
通讯作者：
Hori, Hiroyuki
中科院分区：
文献类型：
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作者：
Awai, Takako;Kimura, Satoshi;Hori, Hiroyuki
转移RNA（N-2，N-2-鸟嘌呤）-二甲基转移酶（Trm 1）催化tRNA中26位（m（2）（2）G26）的N-2，N-2-二甲基鸟嘌呤形成。在该反应中，在26位的N-2-鸟嘌呤（m（2）G26）作为中间体生成。trm 1基因仅在古生菌和真核生物中发现，尽管已经报道超嗜热真细菌Aquifex aeolicus具有推定的trm 1基因。为了确定A. aeolicus Trm 1具有tRNA甲基转移酶活性，我们纯化了重组Trm 1蛋白。体外甲基转移实验表明该蛋白具有较强的tRNA甲基转移酶活性。我们证实了该基因产物在活的A. aeolicus细胞，细胞提取物中存在酶活性。通过制备22个tRNA转录物并测试其甲基接受活性，证明该Trm 1蛋白具有新的tRNA特异性。质谱分析表明，它不仅催化底物tRNA中的甲基转移到G26，还催化甲基转移到G27。此外，证实了天然tRNA（Cys）具有m（2）（2）G26 m（2）G27或m（2）（2）G26 m（2）（2）G27序列，表明这些修饰发生在活细胞中。动力学研究表明，m（2）G26的形成比m（2）G27的形成快，并且G27-C43碱基对的破坏加速了G27修饰的速度。此外，我们制备了另外22个突变体tRNA转录本，并阐明了识别位点存在于T臂结构中。这种长距离识别导致酶的多位点识别。
Transfer RNA (N-2, N-2-guanine)-dimethyltransferase (Trm1) catalyzes N-2, N-2-dimethylguanine formation at position 26 (m(2)(2)G26) in tRNA. In the reaction, N-2-guanine at position 26 (m(2)G26) is generated as an intermediate. The trm1 genes are found only in archaea and eukaryotes, although it has been reported that Aquifex aeolicus, a hyper-thermophilic eubacterium, has a putative trm1 gene. To confirm whether A. aeolicus Trm1 has tRNA methyltransferase activity, we purified recombinant Trm1 protein. In vitro methyl transfer assay revealed that the protein has a strong tRNA methyltransferase activity. We confirmed that this gene product is expressed in living A. aeolicus cells and that the enzymatic activity exists in cell extract. By preparing 22 tRNA transcripts and testing their methyl group acceptance activities, it was demonstrated that this Trm1 protein has a novel tRNA specificity. Mass spectrometry analysis revealed that it catalyzes methyl transfers not only to G26 but also to G27 in substrate tRNA. Furthermore, it was confirmed that native tRNA(Cys) has an m(2)(2)G26m(2)G27 or m(2)(2)G26m(2)(2)G27 sequence, demonstrating that these modifications occur in living cells. Kinetic studies reveal that the m(2)G26 formation is faster than the m(2)G27 formation and that disruption of the G27-C43 base pair accelerates velocity of the G27 modification. Moreover, we prepared an additional 22 mutant tRNA transcripts and clarified that the recognition sites exist in the T-arm structure. This long distance recognition results in multisite recognition by the enzyme.