Biogenesis and growth phase-dependent alteration of 5-methoxycarbonylmethoxyuridine in tRNA anticodons

Nucleic Acids Res. 2016 Jan 29;44(2):509-23. doi: 10.1093/nar/gkv1470. Epub 2015 Dec 17.


Post-transcriptional modifications at the anticodon first (wobble) position of tRNA play critical roles in precise decoding of genetic codes. 5-carboxymethoxyuridine (cmo(5)U) and its methyl ester derivative 5-methoxycarbonylmethoxyuridine (mcmo(5)U) are modified nucleosides found at the anticodon wobble position in several tRNAs from Gram-negative bacteria. cmo(5)U and mcmo(5)U facilitate non-Watson-Crick base pairing with guanosine and pyrimidines at the third positions of codons, thereby expanding decoding capabilities. By mass spectrometric analyses of individual tRNAs and a shotgun approach of total RNA from Escherichia coli, we identified mcmo(5)U as a major modification in tRNA(Ala1), tRNA(Ser1), tRNA(Pro3) and tRNA(Thr4); by contrast, cmo(5)U was present primarily in tRNA(Leu3) and tRNA(Val1). In addition, we discovered 5-methoxycarbonylmethoxy-2'-O-methyluridine (mcmo(5)Um) as a novel but minor modification in tRNA(Ser1). Terminal methylation frequency of mcmo(5)U in tRNA(Pro3) was low (≈30%) in the early log phase of cell growth, gradually increased as growth proceeded and reached nearly 100% in late log and stationary phases. We identified CmoM (previously known as SmtA), an AdoMet-dependent methyltransferase that methylates cmo(5)U to form mcmo(5)U. A luciferase reporter assay based on a +1 frameshift construct revealed that terminal methylation of mcmo(5)U contributes to the decoding ability of tRNA(Ala1).

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Anticodon / chemistry
  • Anticodon / metabolism*
  • Base Pairing
  • Codon / chemistry
  • Codon / metabolism*
  • Escherichia coli / genetics*
  • Escherichia coli / metabolism
  • Gammaproteobacteria / genetics
  • Gammaproteobacteria / metabolism
  • Gene Expression Regulation, Bacterial
  • Genetic Code
  • Models, Molecular
  • Molecular Sequence Data
  • Nucleic Acid Conformation
  • RNA, Bacterial / chemistry
  • RNA, Bacterial / metabolism*
  • RNA, Transfer / chemistry
  • RNA, Transfer / metabolism*
  • Ribosomes / genetics
  • Ribosomes / metabolism
  • Sequence Alignment
  • Uridine / analogs & derivatives
  • Uridine / metabolism*


  • Anticodon
  • Codon
  • RNA, Bacterial
  • RNA, Transfer
  • Uridine