Stable isotope-edited NMR analysis of Ascaris suum mitochondrial tRNAMet having a TV-replacement loop

Abstract

Most nematode mitochondrial (mt) tRNAs have a TV-replacement loop (TV loop) which replaces the normal T arm and the variable loop in standard tRNAs with a less-structured loop. The tertiary structure of such tRNAs has been discussed theoretically with reference to the crystal structure of yeast tRNAPhe [Wolstenholme et al. (1994) Nucleic Acids Res. 22, 4300-4306] and examined experimentally by chemical and enzymatic probing [Watanabe et al. (1994) J. Biol. Chem. 269, 22902-22906]. The results suggest that most regions of the tRNA other than the TV loop are folded in a similar manner to yeast tRNAPhe. To confirm this notion more clearly, the tertiary structure of Ascaris suum mt tRNAMet was analyzed by NMR using various synthetic tRNAs site-specifically labeled with stable isotopes, which were prepared by a combination of chemical synthesis and enzymatic ligation. Tertiary interactions involving G(L2), G(L3), U(L4), and U8 were observed in the NMR spectra of the labeled tRNAs, but those relating to G(L5) were not. On the basis of these results, a possible tertiary structural model of nematode mitochondrial tRNAMet was constructed.