We demonstrate that surface-induced dissociation (SID) coupled with ion mobility-mass spectrometry (IM-MS) is a powerful tool for determining the stoichiometry and quaternary structure of a multi-subunit ribonucleoprotein (RNP) complex assembled in Mg2+. We investigated here Pyrococcus furiosus (Pfu) RNase P, an archaeal RNP that catalyzes tRNA 5′ maturation. Previous step-wise, Mg2+-dependent reconstitutions of Pfu RNase P using its catalytic RNA subunit and two interacting protein cofactor pairs (RPP21•RPP29 and POP5•RPP30) revealed functional RNP intermediates en route to the RNase P enzyme, but provided no information on subunit stoichiometry. Our native MS studies with the proteins alone showed RPP21•RPP29 and (POP5•RPP30)2 complexes, but indicated a 1:1 composition for all subunits when either or both protein complexes bind the cognate RNA. These results highlight the utility of SID and IM-MS in resolving conformational heterogeneity and yielding insights on RNP assembly.

RNase P catalyzes 5'-maturation of tRNAs. While bacterial RNase P comprises an RNA catalyst and a protein cofactor, the eukaryotic (nuclear) variant contains an RNA and up to ten proteins, all unrelated to the bacterial protein. Unexpectedly, a nuclear-encoded bacterial RNase P protein (RPP) homolog is found in several prasinophyte algae including Ostreococcus tauri. We demonstrate that recombinant O. tauri RPP can functionally reconstitute with bacterial RNase P RNAs (RPRs) but not with O. tauri organellar RPRs, despite the latter's presumed bacterial origins. We also show that O. tauri PRORP, a homolog of Arabidopsis PRORP-1, displays tRNA 5'-processing activity in vitro. We discuss the implications of the striking diversity of RNase P in O. tauri, the smallest known free-living eukaryote.

The RNA-binding protein L7Ae, known for its role in translation (as part of ribosomes) and RNA modification (as part of sn/oRNPs), has also been identified as a subunit of archaeal RNase P, a ribonucleoprotein complex that employs an RNA catalyst for the Mg2+-dependent 5′ maturation of tRNAs. To better understand the assembly and catalysis of archaeal RNase P, we used a site-specific hydroxyl radical-mediated footprinting strategy to pinpoint the binding sites of Pyrococcus furiosus (Pfu) L7Ae on its cognate RNase P RNA (RPR). L7Ae derivatives with single-Cys substitutions at residues in the predicted RNA-binding interface (K42C/C71V, R46C/C71V, V95C/C71V) were modified with an iron complex of EDTA-2-aminoethyl 2-pyridyl disulfide. Upon addition of hydrogen peroxide and ascorbate, these L7Ae-tethered nucleases were expected to cleave the RPR at nucleotides proximal to the EDTA-Fe–modified residues. Indeed, footprinting experiments with an enzyme assembled with the Pfu RPR and five protein cofactors (POP5, RPP21, RPP29, RPP30 and L7Ae–EDTA-Fe) revealed specific RNA cleavages, localizing the binding sites of L7Ae to the RPR's catalytic and specificity domains. These results support the presence of two kink-turns, the structural motifs recognized by L7Ae, in distinct functional domains of the RPR and suggest testable mechanisms by which L7Ae contributes to RNase P catalysis.

RNase P is primarily responsible for the 5΄ maturation of transfer RNAs (tRNAs) in all domains of life. Archaeal RNase P is a ribonucleoprotein made up of one catalytic RNA and five protein cofactors including L7Ae, which is known to bind the kink-turn (K-turn), an RNA structural element that causes axial bending. However, the number and location of K-turns in archaeal RNase P RNAs (RPRs) are unclear. As part of an integrated approach, we used native mass spectrometry to assess the number of L7Ae copies that bound the RPR and site-specific hydroxyl radical-mediated footprinting to localize the K-turns. Mutagenesis of each of the putative K-turns singly or in combination decreased the number of bound L7Ae copies, and either eliminated or changed the L7Ae footprint on the mutant RPRs. In addition, our results support an unprecedented ‘double K-turn’ module in type A and type M archaeal RPR variants.

We demonstrate that surface‐induced dissociation (SID) coupled with ion mobility mass spectrometry (IM‐MS) is a powerful tool for determining the stoichiometry of a multi‐subunit ribonucleoprotein (RNP) complex assembled in a solution containing Mg2+. We investigated Pyrococcus furiosus (Pfu) RNase P, an archaeal RNP that catalyzes tRNA 5′ maturation. Previous step‐wise, Mg2+‐dependent reconstitutions of Pfu RNase P with its catalytic RNA subunit and two interacting protein cofactor pairs (RPP21⋅RPP29 and POP5⋅RPP30) revealed functional RNP intermediates en route to the RNase P enzyme, but provided no information on subunit stoichiometry. Our native MS studies with the proteins showed RPP21⋅RPP29 and (POP5⋅RPP30)2 complexes, but indicated a 1:1 composition for all subunits when either one or both protein complexes bind the cognate RNA. These results highlight the utility of SID and IM‐MS in resolving conformational heterogeneity and yielding insights on RNP assembly.