In vitro and in vivo self-cleavage of a viroid RNA with a mutation in the hammerhead catalytic pocket.

Peach latent mosaic viroid (PLMVd) can adopt hammerhead structures in both polarity strands. In the course of a study on the variability of this viroid a natural sequence variant has been characterized in which the hammerhead structure of the plus polarity strand has the sequence CCGA instead of the conserved uridine turn motif CUGA present in the catalytic pocket of all natural hammerhead structures. The viroid RNA containing this mutant hammerhead structure, but not those with the two other possible substitutions, U-->A and U-->G, in the same position of the catalytic pocket, showed significant self-cleavage activity during in vitro transcription. Moreover, the corresponding full-length PLMVd cDNA was infectious and the mutation was retained in a fraction of the viroid progeny. These results indicate that the sequence flexibility of the hammerhead structure, acting in vitro and in vivo , is higher than anticipated and provide relevant data for a deeper insight into the catalytic mechanism of this class of ribozymes and into the structure of the uridine turn motif.