Efficient, pH-Dependent RNA Ligation by the VS Ribozyme in Trans

Publisher Website
Google Scholar
Abstract
The VS ribozyme acts as a very efficient ligase in trans when the 5' cleavage product is prevented from dissociation by an extended helix Ia in the substrate. Provided that the length of this helix is >or=10 bp, the substrate becomes approximately 80% ligated by the ribozyme acting in trans. Most of the nucleotides that have been shown to be important for cleavage are similarly important for ligation, including the critical A756 of the active site. The exception to this is C755. The variant ribozyme C755A has almost normal cleavage activity, whereas the rate of ligation is reduced 70-fold. It is therefore likely that this nucleotide plays a specific role in the organization of the termini of the ligation substrates. We have found that the rate of the trans ligation reaction depends on pH, corresponding to the protonation/deprotonation of a group with a pK(A) of 5.6. A model is suggested whereby the approach to equilibrium is catalyzed by the ribozyme catalyzing the ligation reaction in its deprotonated state (rate 1.05 min(-1)) and the cleavage reaction in its protonated state (rate 0.18 min(-1)). A756 is a candidate for the nucleobase undergoing protonation/deprotonation.