# A minimal RNA substrate with dual fluorescent probes enables rapid kinetics and provides insight into bacterial RNase P active site interactions

## TL;DR

Researchers developed a minimal RNA substrate to study bacterial RNase P, revealing how metal ions and active site mutations affect its catalytic activity.

## Contribution

A new minimal RNA substrate with dual fluorescent probes enables rapid kinetic analysis of bacterial RNase P active site interactions.

## Key findings

- U69 mutations reduced catalysis by over 500-fold in Mg2+ but not in Ca2+.
- CryoEM and SHAPE mapping showed increased flexibility of U69 in Ca2+ compared to Mg2+.
- The study provides tools to analyze RNase P kinetics and active site assembly.

## 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 Mg2+ 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 Ca2+ compared to reactions containing native Mg2+ ions. CryoEM structures and SHAPE mapping suggested increased flexibility of U69 and adjacent nucleotides in Ca2+ compared to Mg2+. These results support a model in which slow catalysis in Ca2+ 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.

Bacterial ribonuclease P (RNase P) is a tRNA processing endonuclease that occurs primarily as a ribonucleoprotein with a catalytic RNA subunit (P RNA).