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Protein-only ribonuclease P (PRORP) is an enzyme responsible for catalyzing the 5′ end maturation of precursor transfer ribonucleic acids (pre-tRNAs) encoded by various cellular compartments in many eukaryotes. PRORPs from plants act as single-subunit enzymes and have been used as a model system for analyzing the function of the metazoan PRORP nuclease subunit, which requires two additional proteins for efficient catalysis. There are currently few molecular details known about the PRORP–pre-tRNA complex. Here, we characterize the determinants of substrate recognition by the single subunit Arabidopsis thaliana PRORP1 and PRORP2 using kinetic and thermodynamic experiments. The salt dependence of binding affinity suggests 4–5 contacts with backbone phosphodiester bonds on substrates, including a single phosphodiester contact with the pre-tRNA 5′ leader, consistent with prior reports of …