Structural characterization of the 69 nucleotide potato spindle tuber viroid left-terminal domain by NMR and thermodynamic analysis

The 69 nucleotide left-terminal domain (TO of the potato spindle tuber RNA viroid (PSTVd) constitutes one of its five structural elements. Due to a twofold complementary sequence repeat, two possible conformations are proposed for the T, secondary structure; an elongated-rod and a bifurcated form. In the present study, two TL mutants were designed that remove the symmetry of the sequence repeats and ensure that either the bifurcated or the elongated-rod conformation is thermodynamically favored. Imino H-1 and N-15 resonances were assigned for both mutants and the native TL domain based on H-1-H-1 NOESY and heteronuclear H-1-N-15 HSQC high-resolution NMR spectra. The NMR secondary structure analysis of all constructs establishes unambiguously the elongated-rod form as the secondary structure of the native TL domain. Temperature-gradient gel electrophoresis and UV melting experiments corroborate these results. A combined secondary structure and sequence analysis of TL domains of other Pospiviroidae family members indicates that the elongated-rod form is thermodynamically favored for the vast majority of these viroids. (C) 2003 Elsevier Ltd. All rights reserved.