Use of fluorescence resonance energy transfer (FRET) to elucidate structure-function relationships in archaeal RNase P, a multi-subunit catalytic ribonucleoprotein

Ribonuclease P (RNase P) is a Mg²⁺-dependent, essential endoribonuclease that catalyzes removal of the 5'-leader of precursor-tRNAs (pre-tRNAs) in all three domains of life. Ribonucleoprotein (RNP) or protein-only variants of RNase P perform this reaction. The RNP form, the focus of this dissertation, consists of one catalytic RNase P RNA (RPR) that is aided by a varying number of RNase P proteins (RPPs): one in bacteria, up to five in archaea, and up to ten in eukaryotes. The experimental tractability and fractional activity of partially assembled complexes make RNase P an ideal model system to investigate protein-aided RNA catalysis, especially the specific gains upon a ribozyme’s accretion of protein cofactors. Archaeal RNase P, whose RPPs are homologous to their eukaryotic counterparts, is an attractive system to study structure-function relationships in a multi-subunit, catalytic RNP. Four of the five archaeal RPPs work as two binary complexes (POP5•RPP30 and RPP21•RPP29), while one RPP (L7Ae) acts independently. These RPPs, separately and together, improve the catalytic efficiency and fidelity of cleavage. Moreover, while ~250-500 mM Mg²⁺ renders the archaeal RPR active without RPPs, addition of all RPPs lowers the Mg²⁺ requirement to ~10-20 mM and helps the RPR function efficiently at near-physiological [Mg²⁺]. Although a cryo-electron microscopy structure of the complete archaeal RNase P complex is available, there are sparse insights into how these RPPs collectively and individually modulate RPR structure for efficient catalysis. Here, I sought to address this gap by using RNase P from Pyrococcus furiosus (Pfu), a hyperthermophilic archaeon.

The RPR is organized into two independently folding modules: the catalytic (C) and specificity (S) domains. To catalyze Mg²⁺-dependent cleavage of the 5'-leader, the two domains cooperate to recognize different parts of the pre-tRNA. The S and C domains accomplish substrate recognition and cleavage, respectively. To understand the Mg²⁺- and RPP-dependent structural changes that increase activity, we used pre-tRNA cleavage and ensemble fluorescence resonance energy transfer (FRET) assays to characterize inter-domain interactions in 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 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. This work also provides a roadmap to obtain dual-labeled RPR and lays the groundwork for future ensemble and single-molecule FRET studies to determine RPR structural changes engendered by different suites of RPPs. Such an undertaking will help map the hierarchy and cooperativity during RNase P assembly. The growing appreciation of diverse roles of RNA/RNPs in cellular processes necessitates a thorough knowledge of their functional alliance. Our studies here provide an experimental framework to explore nuances of protein-mediated broadening of the RNA functional repertoire.