Determination of the Specificity Landscape for Ribonuclease P Processing of Precursor tRNA 5′ Leader Sequences

Maturation of tRNA depends on a single endonuclease, ribonuclease P (RNase P), to remove highly variable 5′ leader sequences from precursor tRNA transcripts. Here, we use high-throughput enzymology to report multiple-turnover and single-turnover kinetics for Escherichia coli RNase P processing of all possible 5′ leader sequences, including nucleotides contacting both the RNA and protein subunits of RNase P. The results reveal that the identity of N(−2) and N(−3) relative to the cleavage site at N(1) primarily control alternative substrate selection and act at the level of association not the cleavage step. As a consequence, the specificity for N(−1), which contacts the active site and contributes to catalysis, is suppressed. This study demonstrates high-throughput RNA enzymology as a means to globally determine RNA specificity landscapes and reveals the mechanism of substrate discrimination by a widespread and essential RNA-processing enzyme.