The Escherichia coli RlmN methyltransferase is a dual-specificity enzyme that modifies both rRNA and tRNA and controls translational accuracy

RNA. 2012 Oct;18(10):1783-95. doi: 10.1261/rna.033266.112. Epub 2012 Aug 13.

Abstract

Modifying RNA enzymes are highly specific for substrate-rRNA or tRNA-and the target position. In Escherichia coli, there are very few multisite acting enzymes, and only one rRNA/tRNA dual-specificity enzyme, pseudouridine synthase RluA, has been identified to date. Among the tRNA-modifying enzymes, the methyltransferase responsible for the m(2)A synthesis at purine 37 in a tRNA set still remains unknown. m(2)A is also present at position 2503 in the peptidyl transferase center of 23S RNA, where it is introduced by RlmN, a radical S-adenosyl-L-methionine (SAM) enzyme. Here, we show that E. coli RlmN is a dual-specificity enzyme that catalyzes methylation of both rRNA and tRNA. The ΔrlmN mutant lacks m(2)A in both RNA types, whereas the expression of recombinant RlmN from a plasmid introduced into this mutant restores tRNA modification. Moreover, RlmN performs m(2)A(37) synthesis in vitro using a tRNA chimera as a substrate. This chimera has also proved useful to characterize some tRNA identity determinants for RlmN and other tRNA modification enzymes. Our data suggest that RlmN works in a late step during tRNA maturation by recognizing a precise 3D structure of tRNA. RlmN inactivation increases the misreading of a UAG stop codon. Since loss of m(2)A(37) from tRNA is expected to produce a hyperaccurate phenotype, we believe that the error-prone phenotype exhibited by the ΔrlmN mutant is due to loss of m(2)A from 23S rRNA and, accordingly, that the m(2)A2503 modification plays a crucial role in the proofreading step occurring at the peptidyl transferase center.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Base Sequence
  • Catalysis
  • Cloning, Molecular
  • Escherichia coli Proteins / metabolism
  • Escherichia coli Proteins / physiology*
  • Methyltransferases / metabolism
  • Methyltransferases / physiology*
  • Models, Biological
  • Models, Molecular
  • Molecular Sequence Data
  • Nucleic Acid Conformation
  • Protein Biosynthesis* / physiology
  • RNA, Ribosomal / chemistry
  • RNA, Ribosomal / genetics
  • RNA, Ribosomal / metabolism*
  • RNA, Transfer / chemistry
  • RNA, Transfer / genetics
  • RNA, Transfer / metabolism*
  • Reproducibility of Results
  • Substrate Specificity

Substances

  • Escherichia coli Proteins
  • RNA, Ribosomal
  • RNA, Transfer
  • Methyltransferases
  • RlmN protein, E coli