Substrate tRNA recognition mechanism of tRNA (m7G46) methyltransferase from Aquifex aeolicus
Substrate tRNA recognition mechanism of tRNA (m7G46) methyltransferase from Aquifex aeolicus
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DOI：
10.1074/jbc.m408209200
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发表时间：
2004-11-19
影响因子：
4.8
通讯作者：
Hori, H
中科院分区：
文献类型：
--
作者：
Okamoto, H;Watanabe, K;Hori, H
转运RNA（m（7）G46）甲基转移酶催化S-腺苷甲硫氨酸甲基转移到tRNA中鸟嘌呤46位的N-7原子上。对风产液囊菌基因组的分析揭示了编码该基因的一个候选开放阅读框aq 065。在大肠杆菌中表达aq 065蛋白，并在15%SDS-聚丙烯酰胺凝胶电泳上纯化至均一。虽然aq 065蛋白的氨基酸序列与大肠杆菌的氨基酸序列有很大的不同。coli YggH中表达，纯化的aq 065蛋白具有tRNA（m（7）G46）甲基转移酶活性。采用液相色谱-质谱联用技术对修饰核苷及其位置进行了测定。为了阐明该酶的RNA识别机制，我们研究了该酶对28种酵母tRNA变体（Phe）和大肠杆菌的甲基转移活性。coli tRNA（Thr）。证实了tRNA前体的5 '-前导RNA和3'-尾随RNA对于甲基转移是不需要的。我们发现，酶的特异性是严重依赖于可变环的大小。使用截短变体的实验表明，插入两个茎之间的可变环序列被识别为底物，并且最重要的识别位点包含在T茎内。这些结果表明，甲基接受活性不需要L形tRNA结构。还发现三维核心中G46周围的核苷酸取代降低了活性。
Transfer RNA (m(7)G46) methyltransferase catalyzes the methyl transfer from S-adenosylmethionine to N-7 atom of the guanine 46 residue in tRNA. Analysis of the Aquifex aeolicus genome revealed one candidate open reading frame, aq065, encoding this gene. The aq065 protein was expressed in Escherichia coli and purified to homogeneity on 15% SDS-polyacrylamide gel electrophoresis. Although the overall amino acid sequence of the aq065 protein differs considerably from that of E. coli YggH, the purified aq065 protein possessed a tRNA (m(7)G46) methyltransferase activity. The modified nucleoside and its location were determined by liquid chromatography-mass spectroscopy. To clarify the RNA recognition mechanism of the enzyme, we investigated the methyl transfer activity to 28 variants of yeast tRNA(Phe) and E. coli tRNA(Thr). It was confirmed that 5'-leader and 3'-trailer RNAs of tRNA precursor are not required for the methyl transfer. We found that the enzyme specificity was critically dependent on the size of the variable loop. Experiments using truncated variants showed that the variable loop sequence inserted between two stems is recognized as a substrate, and the most important recognition site is contained within the T stem. These results indicate that the L-shaped tRNA structure is not required for methyl acceptance activity. It was also found that nucleotide substitutions around G46 in three-dimensional core decrease the activity.