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

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.

Rapid Ribonuclease P Kinetics Measured by Stopped-Flow Fluorescence and Fluorescence Anisotropy.

Stopped-flow fluorescence spectroscopy is a highly sensitive method for measuring rapid enzyme kinetics. A wide range of fluorophores can be employed, and fluorescence and fluorescence polarization can be measured. Thus, binding, conformational changes, and catalysis can, in principle, be measured, making it helpful in probing the entire kinetic landscape of a reaction.

The specificity landscape of bacterial ribonuclease P.

Developing quantitative models of substrate specificity for RNA processing enzymes is a key step toward understanding their biology and guiding applications in biotechnology and biomedicine. Optimally, models to predict relative rate constants for alternative substrates should integrate an understanding of structures of the enzyme bound to "fast" and "slow" substrates, large datasets of rate constants for alternative substrates, and transcriptomic data identifying in vivo processing sites. Such data are either available or emerging for bacterial ribonucleoprotein RNase P a widespread and essential tRNA 5' processing endonuclease, thus making it a valuable model system for investigating principles of biological specificity.

Substrate Specificity and Chemical Mechanism for the Reaction Catalyzed by Glutamine Kinase.

Campylobacter jejuni, a leading cause of gastroenteritis worldwide, has a unique O-methyl phosphoramidate (MeOPN) moiety attached to its capsular polysaccharide. Investigations into the biological role of MeOPN have revealed that it contributes to the pathogenicity of C. jejuni, and this modification is important for the colonization of C.