title: Biogenesis and growth phase-dependent alteration of 5-methoxycarbonylmethoxyuridine in tRNA anticodons.
abstract: 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).
authors.affiliation: Department of Chemistry and Biotechnology, Graduate School of Engineering, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan.
authors.forename: Yusuke
authors.initials: Y
authors.lastname: Sakai
authors.affiliation: Department of Chemistry and Biotechnology, Graduate School of Engineering, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan.
authors.forename: Kenjyo
authors.initials: K
authors.lastname: Miyauchi
authors.affiliation: Department of Chemistry and Biotechnology, Graduate School of Engineering, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan.
authors.forename: Satoshi
authors.initials: S
authors.lastname: Kimura
authors.affiliation: Department of Chemistry and Biotechnology, Graduate School of Engineering, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-8656, Japan ts@chembio.t.u-tokyo.ac.jp.
authors.forename: Tsutomu
authors.initials: T
authors.lastname: Suzuki
mesh_terms: D000595:Amino Acid Sequence; D000926:Anticodon / Q000737:chemistry / Q000378:metabolism*; D020029:Base Pairing; D003062:Codon / Q000737:chemistry / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D020563:Gammaproteobacteria / Q000235:genetics / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial; D005815:Genetic Code; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D012329:RNA, Bacterial / Q000737:chemistry / Q000378:metabolism*; D012343:RNA, Transfer / Q000737:chemistry / Q000378:metabolism*; D012270:Ribosomes / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D014529:Uridine / Q000031:analogs & derivatives / Q000378:metabolism*
chemical_list: D000926:Anticodon; D003062:Codon; D012329:RNA, Bacterial; D012343:RNA, Transfer; D014529:Uridine