The reaction kinetics and three-dimensional architecture of a catalytic RNA

Abstract

The Class I ligase ribozyme was isolated previously from random sequences based on its ability to promote a reaction similar to a single step in RNA polymerization: attack of a primer 3'-hydroxyl on a 5'-triphosphate, with formation of a new 3'-5' bond and release of pyrophosphate. Derivatives have been shown to catalyze general primer extension reactions, making the ligase a useful paradigm for RNA self-replication and RNA polymerase biochemistry as well as RNA catalysis in general. In order to establish the ligase as a model system, we have characterized both the reaction and tertiary architecture of the ribozyme. The reaction kinetics of both multiple- and single-turnover ligation were examined, and from these data minimal kinetic frameworks were constructed. These frameworks provide a basis for the interpretation of future mechanistic work, and suggest strategies by which individual steps in the ligation reaction might be targeted for future improvement. In order to test whether the chemical step of Class I ligation could be further optimized, an in vitro selection was performed under conditions that specifically isolated chemistry. Selected variants had a slightly improved chemical step, and substantially improved Mg++-dependence, such that at 0.5 mM Mg++ a composite improved ligase was 50-fold faster than the parent ribozyme. The tertiary architecture of the ligase was examined using hydroxyl radical probing, which provided a measure of the solvent accessibility at each position in the RNA backbone. In collaboration with another group, these data were used to model the tertiary architecture of the ligase in three dimensions. Finally, the predictive value of the model was.tested and confirmed by photocrosslinking experiments.