Poster abstracts
Poster number 21 submitted by Tien-Hao Chen
Use of chemical modification and mass spectrometry to identify substrate-contacting sites in proteinaceous RNase P, a tRNA processing enzyme
Tien-Hao Chen (Department of Chemistry and Biochemistry and Center for RNA Biology, Ohio State University), Akiko Tanimoto (Department of Chemistry and Biochemistry, Ohio State University), Vicki Wysocki (Department of Chemistry and Biochemistry, Ohio State University), Venkat Gopalan (Department of Chemistry and Biochemistry and Center for RNA Biology, Ohio State University)
Abstract:
RNase P is an endonuclease essential for maturation of all transfer RNAs (tRNAs). Among all enzymes in nature, RNase P occupies a unique place: it can use either an RNA- or a protein-based active site for catalyzing the cleavage of the 5ꞌ-leader from precursor tRNAs (pre-tRNAs). These distinct RNase P variants appear to be the products of parallel evolution. The well-studied RNA-based RNase P uses a specificity domain to recognize the pre-tRNA substrate and a catalytic domain to perform cleavage. Coincidentally, the newly-discovered proteinaceous RNase P (PRORP) also possesses two RNA-processing domains: a pentatricopeptide repeat (PPR) domain and a metallonuclease (NYN) domain. Here, we combined chemical modification of lysines and mass spectrometry to identify putative substrate-contacting residues on PRORP. Site-directed mutagenesis of these candidate contact sites in PRORP, either individually or in pairs, was used to validate the inferences from our chemical footprinting studies. Binding assays helped delineate lysines in the first PPR motif as important for substrate recognition – PRORP K101A and K101A/K109A led to three- and six-fold increase compared to the wild type in the dissociation constants for pre-tRNA binding. On the other hand, pre-tRNA processing assays identified a lysine in the NYN domain as a contributor to cleavage of the scissile phosphodiester linkage, as the cleavage rates of PRORP K439A, K101A/K439A and K109A/K439A decreased two-fold relative to the wild type. These findings indicate that PRORP utilizes two small regions in the PPR and NYN domains, respectively, to bind and cleave the pre-tRNA. Our results also show that protein- and RNA-based forms of RNase P have distinct modules for substrate recognition and cleavage, an unexpected parallel in their mode of action given their independent evolution.
Keywords: RNA-Protein interaction, Mass Spectrometry, PRORP