Dissecting Functional Cooperation among Subunits in a Catalytic Ribonucleoprotein

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Ribonuclease P (RNase P) is an enzyme essential for processing precursor tRNAs (ptRNAs) to their mature forms. The RNase P holoenzyme is a ribonucleoprotein (RNP) comprising one essential catalytic RNase P RNA (RPR) and at least one, four, and nine RNase P proteins (RPPs) in bacteria, archaea and eukarya, respectively. A long-term goal of this research is to understand the dynamic functional interplay among the single catalytic RNA and multiple protein cofactors in archaeal RNase P, a surrogate for its eukaryal relative that has proven biochemically intractable so far. Two specific aims will be pursued. First, since currently known archaeal RPPs were computationally identified based on their homology to eukaryal relatives, experimentally uncovering all subunits associated with native RNase P is necessary. Therefore, RNase P from the thermophilic Thermococcus kodakaraensis and mesophilic Methanococcus maripaludis will be isolated and characterized with respect to their subunit make-up. Specifically, homologous recombination will be exploited to replace a chromosomal RPP gene with an affinity-tagged version thus providing a handle to purify the native RNase P holoenzyme. Second, kinetic and thermodynamic studies on different archaeal RPRs with and without RPPs will be used to dissect the contributions of individual RPPs to substrate binding, cleavage rate, and fidelity of processing. Furthermore, the ability of RPPs to enhance affinity for active-site metal ions will be delineated by using a substrate in which the metal-coordinating oxygen at the RNase P cleavage site is substituted with sulfur. Such an atomic substitution renders the substrate cleavable with Cd2+ and not Mg2+, thus providing a strategy to hone in on metal ions required solely for catalysis (Cd2+) and not RPR folding (Mg2+). These results will be compared with those reported for bacterial RNase P, where a single RPP enables RPR catalysis with near-equal efficiency for different ptRNAs and at physiological Mg2+ concentrations. Such a comparison is expected to furnish insights into whether recruitment of multiple RPPs conferred advantages to the archaeal RNase P holoenzyme. Broader impacts: An overall appreciation of cellular metabolism requires a full understanding of all enzymes that are essential for viability such as RNase P. By furnishing insights into the RNA-protein interplay in RNase P catalysis, results from this project will serve as a model for investigations of similar functional coordination in other cellular RNPs. Moreover, studies on RNase P, a rare remnant of the early metabolism suspected to be dominated by RNA enzymes, will contribute to our understanding of RNAs with functions other than those of information content and offer snapshots of their evolution from the RNA world to the existing protein-dominated cell. Project trainees (undergraduates, graduates, and postdoctoral scholars) will be exposed to fundamental mechanistic questions related to a unique biocatalyst and to modern techniques in biochemistry, enzymology, and molecular biology. Advances from this project will be appropriately integrated into coursework on nucleic acid biochemistry to be offered at The Ohio State University and elsewhere. The PI, as co-director of an NSF REU site program, has been active in recruitment of students from under-represented groups and will seek to increase diversity and promote minority participation in this project. The PI's teaching and research interests have also provided an intellectual platform for developing international outreach activities in the form of short courses and workshops.