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 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.
Baumstark
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Institut für Physikalische Biologie, Heinrich-Heine-Universität Düsseldorf, Düsseldorf, Germany.
Riesner
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D001483:Base Sequence; D002467:Cell Nucleus / Q000378:metabolism; D002474:Cell-Free System; D003198:Computer Simulation; D004586:Electrophoresis / Q000379:methods; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D012313:RNA / Q000096:biosynthesis; D012322:RNA Precursors / Q000378:metabolism; D012323:RNA Processing, Post-Transcriptional; D000079962:RNA, Circular; D012367:RNA, Viral / Q000737:chemistry* / Q000378:metabolism; D011198:Solanum tuberosum / Q000821:virology*; D013347:Subcellular Fractions / Q000378:metabolism; D013379:Substrate Specificity; D014772:Viroids / Q000235:genetics*
D012322:RNA Precursors; D000079962:RNA, Circular; D012367:RNA, Viral; D012313:RNA