title: The substrate specificity of tRNA (m1G37) methyltransferase (TrmD) from Aquifex aeolicus.
abstract: Transfer RNA (m(1)G37) methyltransferase (TrmD) catalyzes methyl-transfer from S-adenosyl-L-methionine to the N(1) atom of G37 in tRNA. In Escherichia coli cells, TrmD methylates tRNA species possessing a G36G37 sequence. It was previously believed that G36 was the positive determinant of TrmD recognition. In the current study, we demonstrate that TrmD from Aquifex aeolicus methylates tRNA transcripts possessing an A36G37 sequence as well as tRNA transcripts possessing a G36G37 sequence. In contrast, tRNA transcripts possessing pyrimidine36G37 were not methylated at all. These substrate specificities were confirmed by an in vitro kinetic assay using 16 tRNA transcripts. The modified nucleoside and the position in yeast tRNA(Phe) transcript were confirmed by LC/MS. Furthermore, nine truncated tRNA molecules were tested to clarify the additional recognition site. Unexpectedly, A. aeolicus TrmD protein efficiently methylated the micro helix corresponding to the anti-codon arm. Because the disruption of the anti-codon stem caused the complete loss of the methyl group acceptance activity, the anti-codon stem is essential for the recognition. Moreover, the existence of the D-arm structure inhibited the activity. Recently, it was reported that E. coli TrmD methylates yeast tRNA(Phe) harboring a sequence A36G37. Thus, recognition of the purine36G37 sequence is probably common to eubacteria TrmD proteins.
authors.affiliation: Department of Applied Chemistry, Faculty of Engineering, Ehime University, Bunkyo 3, Matsuyama 790-8577, Japan.
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Takeda
authors.forename: Takashi
authors.initials: T
authors.lastname: Toyooka
authors.forename: Yoshiho
authors.initials: Y
authors.lastname: Ikeuchi
authors.forename: Shin-ichi
authors.initials: S
authors.lastname: Yokobori
authors.forename: Kan
authors.initials: K
authors.lastname: Okadome
authors.forename: Fuyumi
authors.initials: F
authors.lastname: Takano
authors.forename: Tairo
authors.initials: T
authors.lastname: Oshima
authors.forename: Tsutomu
authors.initials: T
authors.lastname: Suzuki
authors.forename: Yaeta
authors.initials: Y
authors.lastname: Endo
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Hori
mesh_terms: D000595:Amino Acid Sequence; D001419:Bacteria / Q000201:enzymology* / Q000235:genetics; D001483:Base Sequence; D002853:Chromatography, Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D013058:Mass Spectrometry; D008745:Methylation; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D010802:Phylogeny; D012329:RNA, Bacterial / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D012359:tRNA Methyltransferases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D012329:RNA, Bacterial; D011994:Recombinant Proteins; D012359:tRNA Methyltransferases