Determination of relative rate constants for in vitro RNA processing reactions by internal competition

Studies of RNA recognition and catalysis typically involve measurement of rate constants for reactions of individual RNA sequence variants by fitting changes in substrate or product concentration to exponential or linear functions. A complementary approach is determination of relative rate constants by internal competition, which involves quantifying the time-dependent changes in substrate or product ratios in reactions containing multiple substrates. Here, we review approaches for determining relative rate constants by analysis of both substrate and product ratios and illustrate their application using the in vitro processing of precursor transfer RNA (tRNA) by ribonuclease P as a model system. The presence of inactive substrate populations is a common complicating factor in analysis of reactions involving RNA substrates, and approaches for quantitative correction of observed rate constants for these effects are illustrated. These results, together with recent applications in the literature, indicate that internal competition offers an alternate method for analyzing RNA processing kinetics using standard molecular biology methods that directly quantifies substrate specificity and may be extended to a range of applications.