Structure of viroid replicative intermediates: physico-chemical studies on SP6 transcripts of cloned oligomeric potato spindle tuber viroid

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Abstract
The structure and structural transitions of transcripts of cloned oligomeric viroid were studied in physico-chemical experiments and stability calculations. Transcripts of (+) and (−) polarity, from unit up to sixfold length, were synthesized from DNA clones of the potato spindle tuber viroid (PSTV) with the SP6 transcription system. Their structural properties were investigated by optical denaturation curves, high performance liquid chromato-graphy (HPLC), electron microscopy, sedimentation-diffusion equilibrium and velocity sedimentation. Secondary structures of the RNAs and theoretical denaturation curves were calculated using an energy optimization program. The secondary structure of lowest free energy for unit length and oligomeric transcripts is a rod-like structure similar to that of the mature circular viroids. When this structure is used as a model for calculations, there is a large degree of agreement between the theoretical and the experimental denaturation curves. At high temperatures, however, (+) strand transcripts exhibited a transition which was more stable than expected from the calculations or than was known from curves of mature viroids. This transition arises from a rearrangement of the central conserved region of viroids to a helical region of 28 stable base pairs either intermolecularly leading to bimolecular complexes, or intramolecularly giving rise to a branched secondary structure. The rearrangement could be detected by electron microscopy, HPLC, and analytical ultracentrifugation. The helical region serves to divide up the oligomeric (+) strand into structural units which may be recognized by cleavage and ligation enzymes which process the oligomeric intermediates to circular mature viroids.