Elucidation of structure-function relationships in Methanocaldococcus jannaschii RNase P, a multi-subunit catalytic ribonucleoprotein

RNase P is a ribonucleoprotein (RNP) that catalyzes removal of the 5 ' leader from precursor tRNAs in all domains of life. A recent cryo-EM study of Methanocaldococcus jannaschii (Mja) RNase P produced a model at 4.6-angstrom resolution in a dimeric configuration, with each holoenzyme monomer containing one RNase P RNA (RPR) and one copy each of five RNase P proteins (RPPs; POP5, RPP30, RPP21, RPP29, L7Ae). Here, we used native mass spectrometry (MS), mass photometry (MP), and biochemical experiments that (i) validate the oligomeric state of the Mja RNase P holoenzyme in vitro, (ii) find a different stoichiometry for each holoenzyme monomer with up to two copies of L7Ae, and (iii) assess whether both L7Ae copies are necessary for optimal cleavage activity. By mutating all kink-turns in the RPR, we made the discovery that abolishing the canonical L7Ae-RPR interactions was not detrimental for RNase P assembly and function due to the redundancy provided by protein-protein interactions between L7Ae and other RPPs. Our results provide new insights into the architecture and evolution of RNase P, and highlight the utility of native MS and MP in integrated structural biology approaches that seek to augment the information obtained from low/medium-resolution cryo-EM models.

Automated summary

RNase P is a ribonucleoprotein that removes the 5' leader from precursor tRNAs. The study reveals the dimeric configuration of Mja RNase P, with each monomer containing one RPR and five RPPs. It confirms the oligomeric state of Mja RNase P and shows the different stoichiometry of each monomer, with up to two copies of L7Ae. Disrupting the L7Ae-RPR interaction does not affect the assembly and function of RNase P due to redundancy in protein-protein interactions.