title: The catalytic domain of topological knot tRNA methyltransferase (TrmH) discriminates between substrate tRNA and nonsubstrate tRNA via an induced-fit process.
abstract: A conserved guanosine at position 18 (G18) in the D-loop of tRNAs is often modified to 2'-O-methylguanosine (Gm). Formation of Gm18 in eubacterial tRNA is catalyzed by tRNA (Gm18) methyltransferase (TrmH). TrmH enzymes can be divided into two types based on their substrate tRNA specificity. Type I TrmH, including Thermus thermophilus TrmH, can modify all tRNA species, whereas type II TrmH, for example Escherichia coli TrmH, modifies only a subset of tRNA species. Our previous crystal study showed that T. thermophilus TrmH is a class IV S-adenosyl-l-methionine-dependent methyltransferase, which maintains a topological knot structure in the catalytic domain. Because TrmH enzymes have short stretches at the N and C termini instead of a clear RNA binding domain, these stretches are believed to be involved in tRNA recognition. In this study, we demonstrate by site-directed mutagenesis that both N- and C-terminal regions function in tRNA binding. However, in vitro and in vivo chimera protein studies, in which four chimeric proteins of type I and II TrmHs were used, demonstrated that the catalytic domain discriminates substrate tRNAs from nonsubstrate tRNAs. Thus, the N- and C-terminal regions do not function in the substrate tRNA discrimination process. Pre-steady state analysis of complex formation between mutant TrmH proteins and tRNA by stopped-flow fluorescence measurement revealed that the C-terminal region works in the initial binding process, in which nonsubstrate tRNA is not excluded, and that structural movement of the motif 2 region of the catalytic domain in an induced-fit process is involved in substrate tRNA discrimination.
authors.affiliation: From the Department of Materials Science and Biotechnology, Graduate School of Science and Engineering, Ehime University, 3 Bunkyo, Matsuyama, Ehime 790-8577, Japan.
authors.forename: Anna
authors.initials: A
authors.lastname: Ochi
authors.affiliation: the Okazaki Institute for Integrative Bioscience and Institute for Molecular Science, 5-1 Higashiyama, Myodaiji, Okazaki, Aichi 444-8787, Japan.
authors.forename: Koki
authors.initials: K
authors.lastname: Makabe
authors.affiliation: From the Department of Materials Science and Biotechnology, Graduate School of Science and Engineering, Ehime University, 3 Bunkyo, Matsuyama, Ehime 790-8577, Japan.
authors.forename: Ryota
authors.initials: R
authors.lastname: Yamagami
authors.affiliation: From the Department of Materials Science and Biotechnology, Graduate School of Science and Engineering, Ehime University, 3 Bunkyo, Matsuyama, Ehime 790-8577, Japan.
authors.forename: Akira
authors.initials: A
authors.lastname: Hirata
authors.affiliation: the Department of Biochemistry and Molecular Biology, Thomas Jefferson University, Philadelphia, Pennsylvania 19107.
authors.forename: Reiko
authors.initials: R
authors.lastname: Sakaguchi
authors.affiliation: the Department of Biochemistry and Molecular Biology, Thomas Jefferson University, Philadelphia, Pennsylvania 19107.
authors.forename: Ya-Ming
authors.initials: YM
authors.lastname: Hou
authors.affiliation: From the Department of Materials Science and Biotechnology, Graduate School of Science and Engineering, Ehime University, 3 Bunkyo, Matsuyama, Ehime 790-8577, Japan.
authors.forename: Kazunori
authors.initials: K
authors.lastname: Watanabe
authors.affiliation: the Department of Biophysics and Biochemistry, Graduate School of Science, University of Tokyo, 2-11-16 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan, and.
authors.forename: Osamu
authors.initials: O
authors.lastname: Nureki
authors.affiliation: the Okazaki Institute for Integrative Bioscience and Institute for Molecular Science, 5-1 Higashiyama, Myodaiji, Okazaki, Aichi 444-8787, Japan.
authors.forename: Kunihiro
authors.initials: K
authors.lastname: Kuwajima
authors.affiliation: From the Department of Materials Science and Biotechnology, Graduate School of Science and Engineering, Ehime University, 3 Bunkyo, Matsuyama, Ehime 790-8577, Japan,; the Venture Business Laboratory, Ehime University, 3 Bunkyo, Matsuyama, Ehime 790-8577, Japan. Electronic address: hori@eng.ehime-u.ac.jp.
authors.forename: Hiroyuki
authors.initials: H
authors.lastname: Hori
mesh_terms: D020816:Amino Acid Motifs; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D017434:Protein Structure, Tertiary; D012329:RNA, Bacterial / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D012343:RNA, Transfer / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D016963:Thermus thermophilus / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D012359:tRNA Methyltransferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D012329:RNA, Bacterial; D011994:Recombinant Proteins; D012343:RNA, Transfer; D012359:tRNA Methyltransferases
keywords: Pre-steady State Kinetics; Protein Structure; RNA Methylation; RNA Methyltransferase; RNA Modification; RNA-Protein Interaction; RNA-binding Proteins; SPOUT Superfamily; SpoU Family; Transfer RNA (tRNA)