title: Substrate tRNA recognition mechanism of eubacterial tRNA (m1A58) methyltransferase (TrmI).
abstract: TrmI generates N(1)-methyladenosine at position 58 (m(1)A58) in tRNA. The Thermus thermophilus tRNA(Phe) transcript was methylated efficiently by T. thermophilus TrmI, whereas the yeast tRNA(Phe) 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 tRNAGGU(Thr)GGU(Thr) contains C60 instead of U60. The tRNAGGU(Thr) 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 tRNAGGU(Thr) had a low affinity for TrmI than tRNA(Phe). Furthermore, analysis of tRNAGGU(Thr) purified from the trmI gene disruptant strain revealed that the other modifications in tRNA accelerated the formation of m(1)A58 by TrmI. Moreover, nucleoside analysis of tRNAGGU(Thr) from the wild-type strain indicated that less than 50% of tRNAGG(Thr) contained m(1)A58. Thus, the results from the in vitro experiments were confirmed by the in vivo methylation patterns.
authors.affiliation: From the Department of Materials Science and Biotechnology, Graduate School of Science and Engineering, Ehime University, Bunkyo 3, Matsuyama, Ehime 790-8577, Japan and.
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Takuma
authors.affiliation: From the Department of Materials Science and Biotechnology, Graduate School of Science and Engineering, Ehime University, Bunkyo 3, Matsuyama, Ehime 790-8577, Japan and.
authors.forename: Natsumi
authors.initials: N
authors.lastname: Ushio
authors.affiliation: From the Department of Materials Science and Biotechnology, Graduate School of Science and Engineering, Ehime University, Bunkyo 3, Matsuyama, Ehime 790-8577, Japan and.
authors.forename: Masayuki
authors.initials: M
authors.lastname: Minoji
authors.affiliation: From the Department of Materials Science and Biotechnology, Graduate School of Science and Engineering, Ehime University, Bunkyo 3, Matsuyama, Ehime 790-8577, Japan and.
authors.forename: Ai
authors.initials: A
authors.lastname: Kazayama
authors.affiliation: Biomedical Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), 2-4-7 Aomi, Koto-ku, Tokyo 135-0064, Japan.
authors.forename: Naoki
authors.initials: N
authors.lastname: Shigi
authors.affiliation: From the Department of Materials Science and Biotechnology, Graduate School of Science and Engineering, Ehime University, Bunkyo 3, Matsuyama, Ehime 790-8577, Japan and.
authors.forename: Akira
authors.initials: A
authors.lastname: Hirata
authors.affiliation: From the Department of Materials Science and Biotechnology, Graduate School of Science and Engineering, Ehime University, Bunkyo 3, Matsuyama, Ehime 790-8577, Japan and.
authors.forename: Chie
authors.initials: C
authors.lastname: Tomikawa
authors.affiliation: From the Department of Materials Science and Biotechnology, Graduate School of Science and Engineering, Ehime University, Bunkyo 3, Matsuyama, Ehime 790-8577, Japan and.
authors.forename: Anna
authors.initials: A
authors.lastname: Ochi
authors.affiliation: From the Department of Materials Science and Biotechnology, Graduate School of Science and Engineering, Ehime University, Bunkyo 3, Matsuyama, Ehime 790-8577, Japan and hori@eng.ehime-u.ac.jp.
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Hori
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D008745:Methylation; D008958:Models, Molecular; D009154:Mutation; D009690:Nucleic Acid Conformation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D012329:RNA, Bacterial / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012343:RNA, Transfer / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012360:RNA, Transfer, Phe / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012363:RNA, Transfer, Thr / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D016963:Thermus thermophilus / Q000201:enzymology / Q000235:genetics / Q000378:metabolism; D012359:tRNA Methyltransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D012329:RNA, Bacterial; D012360:RNA, Transfer, Phe; D012363:RNA, Transfer, Thr; D012343:RNA, Transfer; D012359:tRNA Methyltransferases
keywords: RNA Methylation; RNA Methyltransferase; RNA Modification; Thermophile; Transfer RNA (tRNA)