Generation of viroid conformational isomers that are stable to incubation with magnesium ions and in a nuclear extract from tomato plants

Abstract

We identified conditions for heating and quick cooling viroid RNAs in the presence of salt which lead to the production of conformational isomers stable to incubation for at least 45 minutes at 30 degrees in the presence of magnesium ions. Elution in 0.3 M NaCl allowed the purification of an electrophoretically slow form of an in vitro transcript carrying a complete copy of the potato spindle tuber viroid RNA sequence. Slow forms of this transcript and of kinase-labeled linear viroid RNA persisted for longer than 20 minutes when incubated with a protein-rich extract prepared from the nuclei of uninfected tomato plants, although both were slowly cleaved by a nuclease present in this extract. The fast form of the transcript was highly resistant to this tomato ribonuclease. The slow form of the transcript was much more susceptible to cleavage by RNase T1 than the fast form of this RNA, suggesting that the reduced gel mobility of the slow forms results from their relatively open structure. The ability to purify viroid conformational isomers from polyacrylamide gels will facilitate biochemical studies aimed at identifying host components interacting with RNAs of the viroid replication complex, which may not all be present in the most thermodynamically favored rodlike structure of mature viroids.