title: Determination of the Specificity Landscape for Ribonuclease P Processing of Precursor tRNA 5' Leader Sequences.
abstract: 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.
authors.affiliation: Department of Biochemistry, Case Western Reserve University School of Medicine , Cleveland, Ohio 44106, United States.
authors.forename: Courtney N
authors.initials: CN
authors.lastname: Niland
authors.affiliation: Department of Biochemistry, Case Western Reserve University School of Medicine , Cleveland, Ohio 44106, United States.
authors.forename: Jing
authors.initials: J
authors.lastname: Zhao
authors.affiliation: Department of Biochemistry, Case Western Reserve University School of Medicine , Cleveland, Ohio 44106, United States.
authors.forename: Hsuan-Chun
authors.initials: HC
authors.lastname: Lin
authors.affiliation: School of Business, CUNY Baruch College , New York, New York 10010, United States.
authors.forename: David R
authors.initials: DR
authors.lastname: Anderson
authors.affiliation: Center for RNA Molecular Biology, Case Western Reserve University School of Medicine , Cleveland, Ohio 44106, United States.
authors.forename: Eckhard
authors.initials: E
authors.lastname: Jankowsky
authors.affiliation: Department of Biochemistry, Case Western Reserve University School of Medicine , Cleveland, Ohio 44106, United States.
authors.forename: Michael E
authors.initials: ME
authors.lastname: Harris
mesh_terms: D007700:Kinetics; D012322:RNA Precursors / Q000378:metabolism*; D012323:RNA Processing, Post-Transcriptional; D043262:Ribonuclease P / Q000378:metabolism*; D013379:Substrate Specificity
chemical_list: D012322:RNA Precursors; D043262:Ribonuclease P