Higher-order structure and thermal instability of bovine mitochondrial tRNASerUGA investigated by proton NMR spectroscopy

Although mammalian mitochondrial serine-specific tRNA with the anti-codon UGA (tRNA(Ser)UGA) appears to possess an almost normal clover-leaf secondary structure, it exhibits and extraordinarily low melting temperature (t(m)). An in vitro tRNA(Ser)UGA transcript without modified nucleosides had an even lower t(m) and slightly less hyperchromicity, but its tertiary structure was apparently very similar to that of the native counterpart judging from its aminoacylation activity and the body of experimental evidence so far obtained for canonical tRNAs. The transcript was therefore used to investigate the higher-order structure and thermal instability of tRNA(Ser)UGA. H-1-NMR analysis of the transcript showed that it takes a nearly L-shaped tertiary structure with similar tertiary base-pairings to those found in yeast tRNA(Phe), which is representative of canonical tRNAs. However, magnesium ion titration revealed that Mg2+ affected the chemical shifts of the tRNA(Ser)UGA transcript differently than those of canonical tRNAs so far studied; the former was less sensitive toward Mg2+, especially in the D-arm region. This observation was confirmed by NMR analysis with paramagnetic manganese ion titration. Hill plots derived from the CD spectral changes caused by titration with Mg2+ suggested that the tRNA(Ser)UGA transcript had fewer Mg2+ binding sites than those of yeast tRNA(Phe) as well as its transcript, a finding that was consistent with the NMR data. We thus surmise that the thermal instability of both the transcript and tRNA(Ser)UGA itself originated from a reduction in the number of the divalent ion binding sites within the tRNA molecule. These results suggest a new type of thermal instability for mitochondrial tRNA. (C) 1998 Academic Press.