The dimerization domain of potato spindle tuber viroid, a possible hallmark for infectious RNA.

Covalently closed circular (+) RNA of the potato spindle tuber viroid (PSTVd) can efficiently dimerize noncovalently upon heating and slow cooling in the presence of monovalent cations or Mg2+. In vitro transcription of subgenomic fragments reveals that the ability to dimerize resides in the "upper strand" of its self-complementary rod-like structure. Nuclease probing of these fragments, namely, molecules spanning either the upper or the lower strand of PSTVd, confirms the existence of the previously proposed hairpins I-III, of which hairpin I might contain noncanonical G.A and A.A base pairs. In addition, the upper and lower (+) strands contain large hairpin loops consisting of stretches rich in either adenosine or uridine. Dimerization of the upper (+) strand results in a nuclease-resistant core encompassing hairpin I and is inhibited by an antisense oligonucleotide spanning the entire hairpin; this palindromic domain thus represents the dimerization site. When upper and lower strands were heated and cooled together, no annealing to a viroid-like duplex of both molecules occurs, only dimerization of the upper strand. Therefore, the dimerization hairpin of viroid RNA represents a unique conformational signal that is homologous to similar regions in the human immunodeficiency virus and other retroviruses.

Secondary structure probing of potato spindle tuber viroid (PSTVd) and sequence comparison with other small pathogenic RNA replicons provides evidence for central non-canonical base-pairs, large A-rich loops, and a terminal branch.

Using PCR and in vitro transcription, linear (non-circular) unit-length (+)strand RNA molecules of a lethal PSTVd variant were produced which are able to initiate typical disease symptoms when inoculated into tomato. Non-denaturing gel electrophoresis shows that these transcripts can adopt the same two conformations as circular PSTVd molecules, namely a fast migrating rod-like and a slowly migrating cruciform structure. The rod-like conformer of two end-labelled transcripts was probed with nucleases and dimethyl sulphate, revealing that in solution its right part is identical to computer prediction. In the left part, however, three unique features could be substantiated. (1) In the central region a UV-cross-linkable loop is closed and thus contains non-canonical base-pairs ("loop E structure"). (2) Three large "pre-melting loops" are present at 25 degrees to 37 degrees C. The structure of the leftmost one, which is A-rich and conserved in most viroids, correlates with pathogenicity. (3) Two small stem-loops instead of an unbranched structure are found at the left terminus. These hairpins can form in all "large" viroids (approximately 300 nucleotides or longer), thus placing the dodecamer conserved among these viroids, GGUUCCUGUGGU, within the upper helix and the branch junction. A large viroid from Iresine lacks one of these hairpins, whereas all "small" viroids (approximately 300 nucleotides or smaller) lack both. In several plant virus satellite RNAs and the newt satellite RNA, the motif GAUUU(U) and dodecamer remnants appear in an equivalent structure comprising two or three hairpins. Using lead- and terbium-induced cleavage of the RNA, metal binding sites were found, mostly in loops. Thus, probing of PSTVd RNA and comparison with other.

Gel dependence of electrophoretic mobilities of double-stranded and viroid RNA and estimation of the contour length of a viroid by gel electrophoresis.

Double-stranded (ds) RNA normally exhibits a lower electrophoretic mobility than dsDNA having the same number of base pairs. This has been attributed to its net charge density that is lower than that of B-form DNA. But we show here that dsRNA runs faster than corresponding DNA in gels containing either > or = 2.5% agarose or > or = 8% acrylamide with high crosslinking (19:1 acrylamide:N,N'-methylenebisacrylamide). However, the relative mobility of dsRNA as compared with DNA, extrapolated to 0% gel (0%T), remains constant (0.90 +/- 0.03) in all systems, in support of the charge density hypothesis. In comparison to dsRNA standards, the potato spindle tuber viroid, a small approximately 70% base-paired rod-like pathogenic RNA, is strongly retarded, presumably because of greater flexibility and/or stable curvature. Depending on the gel system, nonlinear extrapolation to 0% T leads to an apparent contour length of 140-230 bp, whereas 130 +/- 20 bp can be determined from electron micrographs and 123-126 bp from secondary structure modeling. We attribute the variation of the electrophoretic behavior of both dsRNA and viroid RNA to interactions with the gel matrix. Nevertheless, extrapolation of the apparent contour length (in bp dsRNA) determined from low-crosslinked polyacrylamide gels (2.6%C) is comparable to the determination by alternative methods.