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. doi: 10.1074/jbc.M113.485128. Epub 2013 Jul 18.


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).

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Motifs
  • Bacterial Proteins / chemistry*
  • Bacterial Proteins / genetics
  • Bacterial Proteins / metabolism
  • Escherichia coli / genetics
  • Escherichia coli / metabolism
  • Protein Structure, Tertiary
  • RNA, Bacterial / chemistry*
  • RNA, Bacterial / genetics
  • RNA, Bacterial / metabolism
  • RNA, Transfer / chemistry*
  • RNA, Transfer / genetics
  • RNA, Transfer / metabolism
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism
  • Substrate Specificity
  • Thermus thermophilus / chemistry*
  • Thermus thermophilus / genetics
  • Thermus thermophilus / metabolism
  • tRNA Methyltransferases / chemistry*
  • tRNA Methyltransferases / genetics
  • tRNA Methyltransferases / metabolism


  • Bacterial Proteins
  • RNA, Bacterial
  • Recombinant Proteins
  • RNA, Transfer
  • tRNA Methyltransferases