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.

Bovine mitochondrial (mt) phenylalanine tRNA (tRNA(Phe)), which lacks the 'conserved' GG and T psi YCG sequences, was efficiently purified by the selective hybridization method using a solid phase DNA probe. The entire nucleotide sequence of the tRNA, including modified nucleotides, was determined and its higher-order structure was investigated using RNaseT2 and chemical reagents as structural probes. The D and T loop regions as well as the anticodon loop region were accessible to RNaseT2, and the N-3 positions of cytidines present in the D and T loops were easily modified under the native conditions in the presence of 10mM Mg2+. On the other hand, the nucleotides present in the extra loop were protected from the chemical modification under the native conditions. From the results of these probing analyses and a comparison of the sequences of mitochondrial tRNA(Phe) genes from various organisms, it was inferred that bovine mt tRNA(Phe) lacks the D loop/T loop tertiary interactions, but does have the canonical extra loop/D stem interactions, which seem to be the main factor for bovine mt tRNA(Phe) to preserve its L-shaped higher-order structure.