The Kinetics of Ribozyme Cleavage: A Tool to Analyze RNA Folding as a Function of Catalysis

As catalytically active RNAs, ribozymes can be characterized by kinetic measurements similar to classical enzyme kinetics. However, in contrast to standard protein enzymes, for which reactions can usually be started by mixing the enzyme with its substrate, ribozymes are typically self-cleaving. The reaction has to be initiated by folding the RNA into its active conformation. Thus, ribozyme kinetics are influenced by both folding and catalytic components and often enable indirect observation of RNA folding. Here, I describe how to obtain quantitative ribozyme cleavage data via denaturing polyacrylamide gel electrophoresis (PAGE) of radioactively labeled in vitro transcripts and discuss general considerations for subsequent kinetic analysis.