Protein cofactors and substrate influence Mg(2+)-dependent structural changes in the catalytic RNA of archaeal RNase P

The ribonucleoprotein (RNP) form of archaeal RNase P comprises one catalytic RNA and five protein cofactors. To catalyze Mg(2+)-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 Mg(2+) renders the archaeal RPR active without RNase P proteins (RPPs), addition of all RPPs lowers the Mg(2+) requirement to ∼10–20 mM and improves the rate and fidelity of cleavage. To understand the Mg(2+)- 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-RPR(C domain) and Cy5-RPR(S 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 Mg(2+)-dependent paths to converge on similar functional states, and binding of the pre-tRNA by the holoenzyme influences Mg(2+) cooperativity. Our findings highlight how Mg(2+) and proteins in multi-subunit RNPs together favor RNA conformations in a dynamic ensemble for functional gains.