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.
ACS chemical biology