Purification, reconstitution, and mass analysis of archaeal RNase P, a multisubunit ribonucleoprotein enzyme.

The ubiquitous ribonucleoprotein (RNP) form of RNase P catalyzes the Mg2+-dependent cleavage of the 5' leader of precursor-transfer RNAs. The rate and fidelity of the single catalytic RNA subunit in the RNase P RNP is significantly enhanced by association with protein cofactors. While the bacterial RNP exhibits robust activity at near-physiological Mg2+ concentrations with a single essential protein cofactor, archaeal and eukaryotic RNase P are dependent on up to 5 and 10 protein subunits, respectively. Archaeal RNase P-whose proteins share eukaryotic homologs-is an experimentally tractable model for dissecting in a large RNP the roles of multiple proteins that aid an RNA catalyst. We describe protocols to assemble RNase P from Methanococcus maripaludis, a methanogenic archaeon. We present strategies for tag-less purification of four of the five proteins (the tag from the fifth is removed post-purification), an approach that helps reconstitute the RNase P RNP with near-native constituents. We demonstrate the value of native mass spectrometry (MS) in establishing the accurate masses (including native oligomers and modifications) of all six subunits in M. maripaludis RNase P, and the merits of mass photometry (MP) as a complement to native MS for characterizing the oligomeric state of protein complexes. We showcase the value of native MS and MP in revealing time-dependent modifications (e.g., oxidation) and aggregation of protein subunits, thereby providing insights into the decreased function of RNase P assembled with aged preparations of recombinant subunits. Our protocols and cautionary findings are applicable to studies of other cellular RNPs.