tRNA recognition by a bacterial tRNA Xm32 modification enzyme from the SPOUT methyltransferase superfamily

Nucleic Acids Res. 2015 Sep 3;43(15):7489-503. doi: 10.1093/nar/gkv745. Epub 2015 Jul 21.


TrmJ proteins from the SPOUT methyltransferase superfamily are tRNA Xm32 modification enzymes that occur in bacteria and archaea. Unlike archaeal TrmJ, bacterial TrmJ require full-length tRNA molecules as substrates. It remains unknown how bacterial TrmJs recognize substrate tRNAs and specifically catalyze a 2'-O modification at ribose 32. Herein, we demonstrate that all six Escherichia coli (Ec) tRNAs with 2'-O-methylated nucleosides at position 32 are substrates of EcTrmJ, and we show that the elbow region of tRNA, but not the amino acid acceptor stem, is needed for the methylation reaction. Our crystallographic study reveals that full-length EcTrmJ forms an unusual dimer in the asymmetric unit, with both the catalytic SPOUT domain and C-terminal extension forming separate dimeric associations. Based on these findings, we used electrophoretic mobility shift assay, isothermal titration calorimetry and enzymatic methods to identify amino acids within EcTrmJ that are involved in tRNA binding. We found that tRNA recognition by EcTrmJ involves the cooperative influences of conserved residues from both the SPOUT and extensional domains, and that this process is regulated by the flexible hinge region that connects these two domains.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Motifs
  • Escherichia coli / enzymology*
  • Escherichia coli Proteins / chemistry*
  • Escherichia coli Proteins / metabolism
  • Methyltransferases / chemistry*
  • Methyltransferases / metabolism
  • Models, Molecular
  • Nucleic Acid Conformation
  • Protein Binding
  • Protein Structure, Tertiary
  • RNA, Transfer / chemistry
  • RNA, Transfer / metabolism*
  • S-Adenosylhomocysteine / chemistry
  • S-Adenosylhomocysteine / metabolism
  • tRNA Methyltransferases / chemistry*
  • tRNA Methyltransferases / metabolism


  • Escherichia coli Proteins
  • RNA, Transfer
  • S-Adenosylhomocysteine
  • Methyltransferases
  • TrmJ protein, E coli
  • tRNA Methyltransferases

Associated data

  • PDB/3ILK
  • PDB/4CND
  • PDB/4CNE
  • PDB/4XBO