A STABLE HAMMERHEAD STRUCTURE IS NOT REQUIRED FOR ENDONUCLEOLYTIC ACTIVITY OF A RIBOZYME IN-VIVO

Cleavage of a specific target, the mRNA encoding the bacterial neomycin phosphotransferase, by mutant satellite RNA of subterranean clover mottle virus (sSCMoV) ribozymes (Rz) was used to study the role of the hammerhead (Hh) structure in Rz activity in cis and in trans. The bimolecular Rz-target RNA interaction was predicted by computer secondary structure analysis. In vivo, endonucleolytic cleavage was determined in plant protoplasts and compared with in vitro results. Two point mutations within the Hh were studied in detail. A Rz mutant with a point mutation in the most distal nucleotide of the catalytic domain (A14G) showed no endonucleolytic activity in vivo. A second point mutation inside helix II (G11.3C) which destabilizes the helix and, according to thermodynamic calculations, should disrupt the conserved Hh structure, unexpectedly displayed Rz activity in trans in vivo. In vitro, this mutant exhibited an activity similar to the wild-type Rz in cis, but no significant activity in trans. It therefore appears that helix II within the Rz Hh structure is not required in vivo for endonucleolytic activity, nor for stability of the Rz transcript, and that in vitro results are inadequate to predict Rz activity in living cells.