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

The influence of RNA secondary structure on the substrate activity of a longer-than-unit length transcript for processing to circular vlroids 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 vlroids 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.