Fine structure melting of viroids as studied by kinetic methods

The conformational transitions of five viroid species were studied by melting analysis and by fast and slow temperature jump techniques. Experiments with the fast temperature jump technique had to be carried out in 10 mM Na-cacodylate, 0.1 M NaCl, 4 M urea, 1 mM EDTA, pH 6.8. In addition to the highly cooperative main transition (Tm between 46.5 and 49 degrees C for different viroid species [1]) all viroids show at higher temperatures an intermediate transition (Tm approximately equal to 57 degrees C) and a high temperature transition (Tm approximately equal to 68 degrees C). The maximum amplitudes of these transitions amount only to about 1% of that of the main transition. The main transition represents a net dissociation of 78 to 94 base pairs depending on the viroid species. The intermediate transition corresponds to the dissociation of two hairpins with 5-10 base pairs each, and 10-20 nucleotides in the loops. The high temperature transition corresponds to a hairpin of 9 G:C pairs and 1 A:U pair and more than 40 bases in the loop. It is shown that these stable hairpins are not part of the native structure but are newly formed during the main transition. Their formation is responsible for the extraordinary cooperativity observed in the main transition. Hairpins can be correlated to defined sequences of PSTV. Based on these studies, on the sequence of PSTV [2], and on a theoretical treatment [3] a detailed description of the whole mechanism of PSTV denaturation is given.