Alternative Substrate Kinetics of Escherichia coli Ribonuclease P: DETERMINATION OF RELATIVE RATE CONSTANTS BY INTERNAL COMPETITION

Abstract

A single enzyme, ribonuclease P (RNase P), processes the 5' ends of tRNA precursors (ptRNA) in cells and organelles that carry out tRNA biosynthesis. This substrate population includes over 80 different competing ptRNAs in Escherichia coli. Although the reaction kinetics and molecular recognition of a few individual model substrates of bacterial RNase P have been well described, the competitive substrate kinetics of the enzyme are comparatively unexplored. To understand the factors that determine how different ptRNA substrates compete for processing by E. coli RNase P, we compared the steady state reaction kinetics of two ptRNAs that differ at sequences that are contacted by the enzyme. For both ptRNAs, substrate cleavage is fast relative to dissociation. As a consequence, V/K, the rate constant for the reaction at limiting substrate concentrations, reflects the substrate association step for both ptRNAs. Reactions containing two or more ptRNAs follow simple competitive alternative substrate kinetics in which the relative rates of processing are determined by ptRNA concentration and their V/K. The relative V/K values for eight different ptRNAs, which were selected to represent the range of structure variation at sites contacted by RNase P, were determined by internal competition in reactions in which all eight substrates were present simultaneously. The results reveal a relatively narrow range of V/K values, suggesting that rates of ptRNA processing by RNase P are tuned for uniform specificity and consequently optimal coupling to precursor biosynthesis.