A Novel Modified Nucleoside Found at the First Position of the Anticodon of Methionine tRNA from Bovine Liver Mitochondria

Methionine tRNA was purified from bovine liver mitochondria, and its nucleotide sequence was determined. The tRNA possesses only three posttranscriptionally modified nucleosides, two pseudouridines in the anticodon and T stems and a previously unknown nucleoside specified by the gene sequence as cytidine, in the first position of the anticodon. Structure analysis of the anticodon nucleoside by mass spectrometry revealed a molecular mass 28 Da greater than that of cytidine, and unmodified ribose, with substitution at C-5 implied by hydrogen–deuterium exchange experiments. Proton NMR of the intact tRNA showed presence of a formyl moiety, thus leading to the candidate structure 5-formylcytidine (f⁵C), not a previously known compound. The structure assignment was confirmed by chemical synthesis and comparison of data from combined HPLC/mass spectrometry and proton NMR for the natural and synthetic nucleosides. The potential function of f⁵C in the tRNA^Met anticodon is discussed with regard to codon-anticodon interactions.