THE RNA OF BOTH POLARITIES OF THE PEACH LATENT MOSAIC VIROID SELF-CLEAVES IN-VITRO SOLELY BY SINGLE HAMMERHEAD STRUCTURES

Hammerhead self-cleavage of dimeric, monomeric, truncated and mutated transcripts derived from both polarities of the peach latent mosaic vi roid (PLMVd) were characterized. In contrast to some results previousl y published for a very close sequence variant (see ref. 1), these RNAs exhibit a virtually identical self-cleavage during transcription and after purification. By self-cleavage of dimeric transcripts with norma l and mutated hammerhead domains and by complementation experiments, w e show that the cleavage reactions involve only single hammerhead stru ctures. This observation contrasts with the case of avocado sunblotch viroid (ASBVd), the other self-cleaving viroid, whose mechanism involv es mostly double hammerhead structures, whereas single hammerhead clea vage is associated with viroid-like plant satellite RNAs. The differen ce in stability between the native secondary structures adopted by vir oids and the autocatalytic structures, including the hammerhead motif, governs the efficiency of the self-cleavage reaction. The transition between these conformers is the limiting step in catalysis and is rela ted exclusively to the left arm region of PLMVd secondary structure, w hich includes the hammerhead sequences. Most of the mutations between the variant we used and the sequence variant previously published are located in this left arm region, which may explain to a great extent t he differences in their cleavage efficiency. No interactions with long -range sequences contributing to the autocatalytic tertiary structure were revealed in these experiments.