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The catalytic domain of topological knot tRNA methyltransferase (TrmH) discriminates between substrate tRNA and nonsubstrate tRNA via an induced-fit process.

J Biol Chem. 2013 Aug 30;288(35):25562-25574. Epub 2013 Jul 18
Anna Ochi 1 , Koki Makabe 2 , Ryota Yamagami 1 , Akira Hirata 1 , Reiko Sakaguchi 3 , Ya-Ming Hou 3 , Kazunori Watanabe 1 , Osamu Nureki 4 , Kunihiro Kuwajima 2 , Hiroyuki Hori 5
Anna Ochi 1 , Koki Makabe 2 , Ryota Yamagami 1 , Akira Hirata 1 , Reiko Sakaguchi 3 , Ya-Ming Hou 3 , Kazunori Watanabe 1 , Osamu Nureki 4 , Kunihiro Kuwajima 2 , Hiroyuki Hori 5
+ et al

[No authors listed]

Author information
  • 1 From the Department of Materials Science and Biotechnology, Graduate School of Science and Engineering, Ehime University, 3 Bunkyo, Matsuyama, Ehime 790-8577, Japan.
  • 2 the Okazaki Institute for Integrative Bioscience and Institute for Molecular Science, 5-1 Higashiyama, Myodaiji, Okazaki, Aichi 444-8787, Japan.
  • 3 the Department of Biochemistry and Molecular Biology, Thomas Jefferson University, Philadelphia, Pennsylvania 19107.
  • 4 the Department of Biophysics and Biochemistry, Graduate School of Science, University of Tokyo, 2-11-16 Yayoi, Bunkyo-ku, Tokyo 113-0032, Japan, and.
  • 5 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.

摘要


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.

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)