Mitochondrial ribonuclease P structure provides insight into the evolution of catalytic strategies for precursor-tRNA 5' processing

Abstract

Ribonuclease P (RNase P) catalyzes the maturation of the 5' end of tRNA precursors. Typically these enzymes are ribonucleoproteins with a conserved RNA component responsible for catalysis. However, protein-only RNase P (PRORP) enzymes process precursor tRNAs in human mitochondria and in all tRNA-using compartments of Arabidopsis thaliana. PRORP enzymes are nuclear encoded and conserved among many eukaryotes, having evolved recently as yeast mitochondrial genomes encode an RNase P RNA. Here we report the crystal structure of PRORP1 from A. thaliana at 1.75 Å resolution, revealing a prototypical metallonuclease domain tethered to a pentatricopeptide repeat (PPR) domain by a structural zinc-binding domain. The metallonuclease domain is a unique high-resolution structure of a Nedd4-BP1, YacP Nucleases (NYN) domain that is a member of the PIN domain-like fold superfamily, including the FLAP nuclease family. The structural similarity between PRORP1 and the FLAP nuclease family suggests that they evolved from a common ancestor. Biochemical data reveal that conserved aspartate residues in PRORP1 are important for catalytic activity and metal binding and that the PPR domain also enhances activity, likely through an interaction with pre-tRNA. These results provide a foundation for understanding tRNA maturation in organelles. Furthermore, these studies allow for a molecular-level comparison of the catalytic strategies used by the only known naturally evolved protein and RNA-based catalysts that perform the same biological function, pre-tRNA maturation, thereby providing insight into the differences between the prebiotic RNA world and the present protein-dominated world.

Fig. 1.

Crystal structure of PRORP1. (A) Structure of PRORP1 (helices shown in blue, strands shown in yellow). (B) Topology map of secondary structure elements. (C) Cartoon depiction of the PRORP1 domain arrangements: PPR domain (residues 95–292; red), central domain (residues 328–357 and 534–570; yellow), and metallonuclease domain (residues 358–533; blue). (D) Electrostatic surface potential.

Fig. 2.

PPR domain architecture and structural Zn-binding site. (A) Tandem repeats of the PPR domain. Domain and profile analysis of PRORP1 using PROSITE predicts three PPR motifs (PPR1, residues 96–130; PPR3, residues 175–209; and PPR4, residues 210–244) on the basis of sequence consensus. The structure reveals two additional helix-turn-helix hairpins that adopt the PPR motif (PPR2, residues 131–174; and PPR5, residues 245–278) despite their divergence from the canonical PPR sequence. The PPR2 hairpin contains an extended loop, ∼11 aa vs. the typically observed 3-aa loop, between α-helices 3 and 4. (B) Structural superposition of PPR/TPR domains: PRORP1 (red; residues 95–293), mtRNAP (yellow; residues 258–331), Get4 (blue; residues 58–236), and the proteasomal subunit Rpn6 (green; residues 38–222). (C) Close-up of the Zn-binding site of PRORP1.

Fig. 3.

Active site, metal dependence, and pre-tRNA binding of PRORP1. (A) Close-up of the Mn(II)-bound PRORP1 active site indicating conserved residues involved in metal binding. (B) Close-up of the Ca(II)-bound PRORP1 active site. (C) Structural alignment of the active sites of the PRORP1 metallonuclease domain (blue) and hExo1 (green bound to DNA) (20). Active-site Mn(II) metals are shown in purple spheres for PRORP1 and in green spheres for hExo1. Black arrows indicate the location of the two aspartates in hExo1 that coordinate the second active-site metal [Mn(II)₂]; there are no equivalent residues in PRORP1 that bind Mn(II)₂. (D) Electron density maps of the active site in the Mn(II)-bound PRORP1. The anomalous difference 2Fo-Fc electron density map, contoured at 3σ, is shown in yellow and is superimposed on the PRORP1 structure. This map was calculated from experimental phases derived from data collected at the Mn edge. The composite omit 2Fo-Fc difference density map for PRORP1 with Mn(II) contoured at 1.5σ is shown in blue. The omit Fo-Fc difference map for the PRORP1 model refined without the active-site Mn atoms and inner sphere water molecules is contoured at 10σ and is shown in green. (E) Representative gels of metal-dependent single-turnover cleavage assays (Upper Left and Lower Left). Reactions containing A. thaliana mitochondrial 5′-³²P-pre-tRNA^Cys, 500 nM Δ76PRORP1, 2.5 mM MgCl₂ or CaCl₂, and 250 μM cobalt(III)hexammine were quenched at specified time points, resolved by denaturing PAGE, and analyzed with a phosphorimager. Mg(II) and Mn(II) activate catalysis of phosphodiester bond hydrolysis whereas Zn(II) and Ca(II) do not (Fig. S2). Fluorescent polarization binding data in 1 mM CaCl₂ (Upper Right) indicate that the binding affinity for fluorescein-labeled mitochondria pre-tRNA^Cys is decreased 34-fold by deletion of four PPR motifs (Δ245PRORP1, blue squares; Δ76PRORP1, black circles). (Lower Right) Cleavage rate constants and K_D values for mutant PRORP1 proteins are summarized. The errors reported for the K_D and k_obs values represent the SD from two and four, respectively, independent experiments (Fig. S3).

Fig. 4.

Mechanistic comparison of the RNA- and protein-based RNase P-catalyzed reaction. (A) Proposed mechanism of cleavage catalyzed by RNA-based RNase P (recreated from ref. 5). The bound metals (M1 and M2) are proposed to be coordinated by nonbridging phosphodiester oxygens and oxygen atoms of nucleotide bases within the catalytic RNA component. The metal (M1)-bound hydroxide is proposed as the catalytic nucleophile. M1 is also proposed to coordinate the pro-R_p oxygen of the scissile phosphate bond. M2 is proposed to position a water molecule for leaving-group stabilization through protonation of the 3′ hydroxyl. (B) Proposed mechanism of cleavage catalyzed by PRORP enzymes. An active-site aspartate is proposed to function as a general base, catalyzing deprotonation of a metal (M1)-bound water to activate the nucleophilic water. On the basis of comparison with hExo1 and lack of a phosphorothioate effect on the pro-R_p oxygen (34), the pro-S_p oxygen of the scissile phosphodiester is predicted to be coordinated by both active-site metal ions to increase electrophilicity and stabilize the transition state. An active-site general acid is proposed to protonate and stabilize the leaving group.