SMALLER, FASTER RIBOZYMES REVEAL THE CATALYTIC CORE OF NEUROSPORA VS RNA

We have investigated the structural requirements for cis-cleavage of t he VS ribozyme by designing deletions, substitutions, and circular per mutations based on the secondary structure model. Four of the six heli ces predicted in the model have been shortened, resulting in self-clea ving RNAs of only 121 to 126 nucleotides. Remarkably, the shorter ribo zymes exhibit a 30 to 40-fold faster cis-cleavage rate. The increase i n activity results from disrupting an inhibitory helix whose 5' side c ontains bases upstream of the cleavage site, and from constructing a c ircular permutation that tethers the helix containing the cleavage sit e to a shortened version of the rest of the ribozyme. The non-essentia l regions identified by the deletions map to the periphery of a recent ly proposed structure model, revealing a central ribozyme core that co ntains the essential structural elements required for activity of the VS ribozyme. (C) 1998 Academic Press Limited.