title: Use of chemical modification and mass spectrometry to identify substrate-contacting sites in proteinaceous RNase P, a tRNA processing enzyme.
abstract: Among all enzymes in nature, RNase P is unique in that it can use either an RNA- or a protein-based active site for its function: catalyzing cleavage of the 5'-leader from precursor tRNAs (pre-tRNAs). The well-studied catalytic RNase P RNA uses a specificity module to recognize the pre-tRNA and a catalytic module to perform cleavage. Similarly, the recently discovered proteinaceous RNase P (PRORP) possesses two domains - pentatricopeptide repeat (PPR) and metallonuclease (NYN) - that are present in some other RNA processing factors. Here, we combined chemical modification of lysines and multiple-reaction monitoring mass spectrometry to identify putative substrate-contacting residues in Arabidopsis thaliana PRORP1 (AtPRORP1), and subsequently validated these candidate sites by site-directed mutagenesis. Using biochemical studies to characterize the wild-type (WT) and mutant derivatives, we found that AtPRORP1 exploits specific lysines strategically positioned at the tips of it's V-shaped arms, in the first PPR motif and in the NYN domain proximal to the catalytic center, to bind and cleave pre-tRNA. Our results confirm that the protein- and RNA-based forms of RNase P have distinct modules for substrate recognition and cleavage, an unanticipated parallel in their mode of action.
authors.affiliation: Department of Chemistry & Biochemistry, The Ohio State University, Columbus, OH 43210, USA Center for RNA Biology, The Ohio State University, Columbus, OH 43210, USA.
authors.forename: Tien-Hao
authors.initials: TH
authors.lastname: Chen
authors.affiliation: Department of Chemistry & Biochemistry, The Ohio State University, Columbus, OH 43210, USA.
authors.forename: Akiko
authors.initials: A
authors.lastname: Tanimoto
authors.affiliation: College of Pharmacy, The Ohio State University, Columbus, OH 43210, USA.
authors.forename: Nikoloz
authors.initials: N
authors.lastname: Shkriabai
authors.affiliation: College of Pharmacy, The Ohio State University, Columbus, OH 43210, USA.
authors.forename: Mamuka
authors.initials: M
authors.lastname: Kvaratskhelia
authors.affiliation: Department of Chemistry & Biochemistry, The Ohio State University, Columbus, OH 43210, USA wysocki.11@osu.edu.
authors.forename: Vicki
authors.initials: V
authors.lastname: Wysocki
authors.affiliation: Department of Chemistry & Biochemistry, The Ohio State University, Columbus, OH 43210, USA Center for RNA Biology, The Ohio State University, Columbus, OH 43210, USA gopalan.5@osu.edu.
authors.forename: Venkat
authors.initials: V
authors.lastname: Gopalan
mesh_terms: D001665:Binding Sites; D020134:Catalytic Domain; D013058:Mass Spectrometry; D008968:Molecular Conformation; D011485:Protein Binding; D012343:RNA, Transfer / Q000737:chemistry / Q000378:metabolism*; D043262:Ribonuclease P / Q000737:chemistry* / Q000378:metabolism*; D013379:Substrate Specificity
chemical_list: D012343:RNA, Transfer; D043262:Ribonuclease P