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RNA modification is one of the most fundamental biological phenomena because it is essential for cell viability through the regulation of protein synthesis. At present, 107 types of RNA modifications are reported. Among them, N2, N2-dimethylguanosine at position 26 (m2 2G26) is found in a half of tRNA species from archaea and 2/3 of tRNA species from eukaryotes. This modification is brought by tRNA (m2 2G26) methyltransferase. This enzyme catalyzes the methyl-transfers from S-adenosyl-L-methionine to N2 atom of the G26. In the reaction, two molecules of S-adenosyl-L-metnionine are consumed and N2-methylguanine at position 26 (m2G26) is generated as an intermediate. A hyper-thermophilic eubacterium, Aquifex aeolicus is supposed to evolutionarily diverge at the earliest period from the other bacteria as judged by the phylogenetic analyses of small rRNA and translation factors. In 1998, genome sequencing project has been reported that a putative trm1 gene is encoded in A. aeolicus genome. In general, the trm1 gene encodes tRNA (m2 2G26) methyltransferase. At present, trm1 gene is not discovered from eubacteria except for A. aeolicus. To clarify whether this putative trm1 gene is encoded tRNA (m2 2G26) methyltransferase or not, we constructed Trm1 overexpression system in E. coli and demonstrated that this trm1 gene product possesses a tRNA (m2 2G26) methyltransferase activity. In this meeting, we discuss the substrate RNA specificity of the A. aeolicus Trm1.