The Escherichia coli RlmN methyltransferase is a dual-specificity enzyme that modifies both rRNA and tRNA and controls translational accuracy.
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.
Laboratorio de Genética Molecular, Centro de Investigación Príncipe Felipe, 46012 Valencia, Spain.
Alfonso
A
Benítez-Páez
Magda
M
Villarroya
M-Eugenia
ME
Armengod
D001483:Base Sequence; D002384:Catalysis; D003001:Cloning, Molecular; D029968:Escherichia coli Proteins / Q000378:metabolism / Q000502:physiology*; D008780:Methyltransferases / Q000378:metabolism / Q000502:physiology*; D008954:Models, Biological; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D014176:Protein Biosynthesis / Q000502:physiology; D012335:RNA, Ribosomal / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D012343:RNA, Transfer / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015203:Reproducibility of Results; D013379:Substrate Specificity
D029968:Escherichia coli Proteins; D012335:RNA, Ribosomal; D012343:RNA, Transfer; D008780:Methyltransferases; C558134:RlmN protein, E coli