Poster abstracts

Poster number 43 submitted by Tien-Hao Chen

Mass spectrometry-aided identification of substrate contact sites in a proteinaceous RNase P

Tien-Hao Chen (Chemistry and Biochemistry), Akiko Tanimoto (Chemistry and Biochemistry), Vicki Wysocki (Chemistry and Biochemistry), Venkat Gopalan (Chemistry and Biochemistry)

Abstract:
RNase P, which catalyzes tRNA 5’-maturation, is an essential enzyme that displays diversity in its make-up: a catalytic RNA-based ribonucleoprotein (RNP) form in all domains of life, and an RNA-free, protein-based version present only in certain eukaryotes. Because RNase P is unique in its ability to use either an RNA- or a protein-based active site for catalyzing the same reaction, understanding substrate recognition and catalysis by these variants might provide insights into the evolution of biocatalysts. Unlike the well-studied RNP form, the proteinaceous RNase P (PRORP) has drawn attention only recently. The crystal structure of PRORP reveals two motifs implicated in RNA processing: pentatricopeptide repeat (PPR) and metallonuclease (MN) domains. The goal of this study is to understand how PRORP uses these two domains to recognize and cleave a precursor tRNA (pre-tRNA) substrate. To identify the substrate-contacting residues, we incubated either the PRORP─pre-tRNA complex or PRORP alone with N-hydroxysuccinimide-biotin to modify solvent-exposed lysines, with the expectation that lysines contacting the pre-tRNA would be protected from modification. The differential accessibility of lysines was determined using proteolysis and mass spectrometry (MS). Our results revealed 86% of the lysines in PRORP are modified. Of these, we identified four pre-tRNA-contacting lysines in the PPR domain, and three in the MN domain. Mutating these lysine residues in either the PPR or MN domains resulted in binding or catalytic defects, respectively, and validated the MS-aided footprinting data. These results lead to two inferences about RNase P diversity and evolution. First, unlike the RNP, PRORP uses a smaller surface area to recognize pre-tRNAs, partly accounting for differences in catalytic potential between these variants. Second, both forms of RNase P appear to have evolutionarily converged on the theme of using distinct domains for substrate recognition and cleavage.

Keywords: RNA-Protein interaction, Mass Spectrometry, PRORP