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

Publisher: Oxford University Press Country of Publication: England NLM ID: 0411011 Publication Model: Print Cited Medium: Internet ISSN: 1362-4962 (Electronic) Linking ISSN: 03051048 NLM ISO Abbreviation: Nucleic Acids Res Subsets: MEDLINE

Publication: 1992- : Oxford : Oxford University Press
Original Publication: London, Information Retrieval ltd.

Archaeal Proteins*/metabolism ; Methanocaldococcus*/enzymology ; Methanocaldococcus*/genetics ; Ribonuclease P*/metabolism; Protein Conformation ; RNA, Transfer/metabolism ; Structure-Activity Relationship

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-Å 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.
(© The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research.)

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R01 GM067807 United States GM NIGMS NIH HHS; S10 OD023582 United States OD NIH HHS; R01 GM122432 United States GM NIGMS NIH HHS; P30 CA016058 United States CA NCI NIH HHS; P41 GM128577 United States GM NIGMS NIH HHS

0 (Archaeal Proteins)
9014-25-9 (RNA, Transfer)
EC 3.1.26.5 (Ribonuclease P)

Date Created: 20220718 Date Completed: 20220815 Latest Revision: 20241107

20241107

PMC9371926

10.1093/nar/gkac595

35848927