Protein cofactors and substrate influence Mg2+-dependent structural changes in the catalytic RNA of archaeal RNase P

Abstract

The ribonucleoprotein (RNP) form of archaeal RNase P comprises one catalytic RNA and five protein cofactors. To catalyze Mg2+-dependent cleavage of the 5′ leader from pre-tRNAs, the catalytic (C) and specificity (S) domains of the RNase P RNA (RPR) cooperate to recognize different parts of the pre-tRNA. While ∼250–500 mM Mg2+ renders the archaeal RPR active without RNase P proteins (RPPs), addition of all RPPs lowers the Mg2+ requirement to ∼10–20 mM and improves the rate and fidelity of cleavage. To understand the Mg2+- and RPP-dependent structural changes that increase activity, we used pre-tRNA cleavage and ensemble FRET assays to characterize inter-domain interactions in Pyrococcus furiosus (Pfu) RPR, either alone or with RPPs ± pre-tRNA. Following splint ligation to doubly label the RPR (Cy3-RPRC domain and Cy5-RPRS domain), we used native mass spectrometry to verify the final product. We found that FRET correlates closely with activity, the Pfu RPR and RNase P holoenzyme (RPR + 5 RPPs) traverse different Mg2+-dependent paths to converge on similar functional states, and binding of the pre-tRNA by the holoenzyme influences Mg2+ cooperativity. Our findings highlight how Mg2+ and proteins in multi-subunit RNPs together favor RNA conformations in a dynamic ensemble for functional gains.

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

“…Because the Mja POP5•RPP30 alone exists as a heterotetramer, it appeared that this complex is key to formation of the holoenzyme dimer (Supplementary Figure S13). But this expectation contrasts with what we previously observed with Pfu (type A) RNase P (47,48). When Pfu RPR was assembled with Pfu POP5•RPP30 in 6 mM Mg 2+ , we observed a complex of only 1 RPR and 1 POP5 + 1 RPP30, even though Pfu POP5•RPP30 alone formed a heterotetramer (47).…”

“…However, with the available resolution, it cannot be determined if this mass shift is due either to formation of adducts with cations or to non-templated addition of nucleotides to the 3 end (49,50). Magnesium adduction seems likely because Mg 2+ is present in the IVT reaction and is required for folding and activity of the RPR (48). For the Mja L7Ae sample, a monomer was observed as expected (Supplementary Figure S3).…”

“…The dimeric configuration found in the cryo-EM structure of Mja (type M) RNase P has not been observed for any archaeal RNase P. When the stoichiometry of Pyrococcus furiosus (Pfu; type A) RPR with Pfu POP5•RPP30 and/or Pfu RPP21•RPP29 was previously investigated by native MS, only a monomeric assembly was detected (47,48). While this difference in the quaternary arrangement of type A versus M archaeal RNase P suggests structural variability in an ancient enzyme, this dissimilarity may have resulted from comparing gas phase versus vitreous state studies or from other technical reasons (e.g.…”

“…While native MS provided accurate information on the composition of Mja RNase P, it is possible that the relative abundance of the observed species may be biased by differences in their ionization efficiency and ion transmission efficiency. Therefore, to better understand the abundance and possible concentration dependence of the Mja RNase P dimer species in solution, we turned to mass photometry (MP; Figure 4 and Supplementary Table S2), which has been used effectively to determine the oligomeric state of proteins, either free or complexed to other macromolecular species (45,48,51,52).…”

Short Oligonucleotides Facilitate Co-transcriptional Labeling of RNA at Specific Positions

“…RNAs are naturally non-fluorescent molecules, and fluorescent labeling RNAs at specific sites is a prerequisite for visualization and investigation of RNA by biophysical techniques, including steady-state fluorescence, FRET, and stopped-flow fluorescent kinetics. − SARS-CoV-2 causes a severe acute respiratory syndrome, and this virus has been a global health emergency since 2019. PRF from SARS-CoV-2 plays essential roles in altering the open reading frame (ORF) of the ribosome, affecting virus particle assembly and virulence release and, therefore, can be developed into a potential drug target. − The PRF sequence used here contains 102 nt, with a pseudoknot formed in the presence of Mg 2+ . , We introduced 2AP at sites 91 and 93 around the pseudoknot via SMRITI (Figures A, A, and S7) and monitored Mg 2+ -derived structural change at these sites by steady-fluorescent titrations (Figure B).…”