Abstract
Bacterial ribonuclease P (RNase P) is a tRNA processing endonuclease that occurs primarily as a ribonucleoprotein with a catalytic RNA subunit (P RNA). As one of the first ribozymes discovered, P RNA is a well-studied model system for understanding RNA catalysis and substrate recognition. Extensive structural and biochemical studies have revealed the structure of RNase P bound to precursor tRNA (ptRNA) and product tRNA. These studies also helped to define active site residues and propose the molecular interactions that are involved in substrate binding and catalysis. However, a detailed quantitative model of the reaction cycle that includes the structures of intermediates and the process of positioning active site metal ions for catalysis is lacking. To further this goal, we used a chemically modified minimal RNA duplex substrate (MD1) to establish a kinetic framework for measuring the functional effects of P RNA active site mutations. Substitution of U69, a critical nucleotide involved in active site Mg(2+) binding, was found to reduce catalysis >500-fold as expected, but had no measurable effect on ptRNA binding kinetics. In contrast, the same U69 mutations had little effect on catalysis in Ca(2+) compared to reactions containing native Mg(2+) ions. CryoEM structures and SHAPE mapping suggested increased flexibility of U69 and adjacent nucleotides in Ca(2+) compared to Mg(2+). These results support a model in which slow catalysis in Ca(2+) is due to inability to engage U69. These studies establish a set of experimental tools to analyze RNase P kinetics and mechanism and can be expanded to gain new insights into the assembly of the active RNase P-ptRNA complex.