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The influence of RNA secondary structure on the substrate activity of a longer-than-unit length transcript for processing to circular viroids was studied in a nuclear extract from potato suspension cells. The nuclear extract was prepared according to a modified procedure for a plant transcription extract. The transcript of the potato spindle tuber viroid (PSTVd) consists of a monomeric molecule with 17 additional nucleotides, thus doubling most of the central conserved region of viroids of the PSTVd-class. The transcript can assume four different secondary structures, which either co-exist as conformers in solution or can be kept as metastable structures after different treatments by temperature and/or ionic strength. The structures were analysed by thermodynamic calculations and temperature-gradient gel electrophoresis and were confirmed by oligonucleotide mapping. Only the so-called extended middle structure was processed to exact viroid circles. In this structure the 5'- and 3'-ends are branching out from the rod-like viroid structure at the loop starting with nucleotide 87. The other structures were processed only if they could be rearranged into the active structure.

nucleic acids research

Only one of four possible secondary structures of the central conserved region of potato spindle tuber viroid is a substrate for processing in a potato nuclear extract