Influence of substrate structure on cleavage by hammerhead ribozyme.

We compared the cleavage by a hammerhead ribozyme of a wild-type precursor tRNA (pre-tRNA leu(3)) and a structurally altered mutant form. We also analyzed the cleavage reactions of these tRNAs catalyzed by a ribozyme variant that was designed to complement the mutant precursor tRNA. Kinetic analyses reveal that the kcat values are nearly the same for the wild-type and the mutant substrate RNAs. However, the Km values differ considerably, being higher for the wild-type substrate. Thus, the formation of the ribozyme-substrate complex, but not the chemical cleavage step, is affected by these changes. Time course studies were performed, at different temperatures, to estimate the efficiency of the cleavage reactions and the effect of temperature. The cleavage of mutant precursor tRNA is generally faster than the wild-type at all temperatures analyzed. These results suggest that substrate structures can limit ribozyme efficiency, presumably by hindering the hybridization step.