Pentatricopeptide repeats of protein-only RNase P use a distinct mode to recognize conserved bases and structural elements of pre-tRNA

Abstract

Pentatricopeptide repeat (PPR) motifs are α-helical structures known for their modular recognition of single-stranded RNA sequences with each motif in a tandem array binding to a single nucleotide. Protein-only RNase P 1 (PRORP1) in Arabidopsis thaliana is an endoribonuclease that uses its PPR domain to recognize precursor tRNAs (pre-tRNAs) as it catalyzes removal of the 5'-leader sequence from pre-tRNAs with its NYN metallonuclease domain. To gain insight into the mechanism by which PRORP1 recognizes tRNA, we determined a crystal structure of the PPR domain in complex with yeast tRNAPhe at 2.85 Å resolution. The PPR domain of PRORP1 bound to the structurally conserved elbow of tRNA and recognized conserved structural features of tRNAs using mechanisms that are different from the established single-stranded RNA recognition mode of PPR motifs. The PRORP1 PPR domain-tRNAPhe structure revealed a conformational change of the PPR domain upon tRNA binding and moreover demonstrated the need for pronounced overall flexibility in the PRORP1 enzyme conformation for substrate recognition and catalysis. The PRORP1 PPR motifs have evolved strategies for protein-tRNA interaction analogous to tRNA recognition by the RNA component of ribonucleoprotein RNase P and other catalytic RNAs, indicating convergence on a common solution for tRNA substrate recognition.

Figure 1.

The PRORP1 PPR domain recognizes the tRNA D and TψC loops. (A) Ribbon diagram of the crystal structure of the PRORP1 PPR-tRNA^Phe complex. PRORP1 PPR is shown in green with tRNA-interacting residues displayed as stick models. The tRNA is shown as a cartoon colored by region: acceptor stem (cyan), D stem loop (blue), anticodon stem loop (magenta), variable region (yellow), and TψC stem loop (orange). (B) Close-up view of the G19–C56 base pair accommodation pocket of the PRORP1 PPR domain. PRORP1 and tRNA are colored as in (A) with atom colors: oxygen (red), nitrogen (blue), phosphorus (orange) and sulfur (yellow). Dashed lines indicate interactions between PRORP1 and tRNA, and the G19–C56 and G18–ψ55 base pairs are indicated by transparent spheres.

Figure 2.

PRORP1 PPR–tRNA interactions. Schematic representation of interactions between the PRORP1 PPR domain and yeast tRNA^Phe. PRORP1 PPR residues are green, tRNA TψC-loop nucleotides are orange, and tRNA D-loop nucleotides are light blue. Circles represent tRNA phosphate groups (P). Dotted and double lines indicate hydrophilic and stacking interactions, respectively.

Figure 3.

PRORP1 PPR motifs use distinct mechanisms for tRNA recognition. (A) Recognition of ψ55 by PRORP1 repeat 3. (B) Recognition of U2 by PPR10 repeat 2 (PDB ID: 4M59). (C) Recognition of G19 by PRORP1 repeat 2 (PDB ID: 6LVR). (D) Differences in specific RNA recognition by PRORP1 and PPR10. RNA-interacting residues in PPR motifs 1–4 of PRORP1 and PPR10 are shown. Nucleotides recognized are shown below and connected by a yellow line. Individual recognition modules are boxed with PRORP1 colored green and PPR10 colored blue.

Figure 4.

Fluorescence anisotropy binding curves for PRORP1 variants binding to B. subtilis 5′-fluorescein-pre-tRNA^Asp substrate. (A) Binding curves for PRORP1 Y133 and R212 variants. (B) Binding curves for PRORP1 R210 variants. WT binding curves are shown in black. The assays were carried out in 30 mM MOPS, pH 7.8, 330 mM NaCl, 1 mM TCEP and 20 mM CaCl₂ at 25 ± 1°C. A hyperbola (Equation 1, Materials and Methods) was fit to the fraction change in anisotropy measured from a single experimental trial using GraphPad Prism to derive values for the dissociation constant (K_D) (Table 2).

Figure 5.

Time courses for the single-turnover cleavage of 5′-fluorescein pre-tRNA substrate catalyzed by PRORP1 variants, measured in 30 mM MOPS, pH 7.8, 330 mM NaCl, 1 mM TCEP and 20 mM MgCl₂ at 25 ± 1°C. The enzyme concentration was 30 μM. A single exponential (Equation 2, Materials and Methods) was fit to the time dependence of the fraction of product formation measured from a single experimental trial using GraphPad Prism to determine a single-turnover rate constant (k_obs) (Table 2). The R212K mutant was evaluated under sub-saturating enzyme concentration. Representative data are shown in Supplementary Figure S10.

Figure 6.

Evolutionary convergence of G19–C56 tRNA base pair recognition. (A) PRORP1 PPR domain recognition of the G19–C56 base pair. (B) Bacterial RNP RNase P RNA recognition of the G19–C56 base pair (PDB ID: 3Q1Q). (C) 23S rRNA recognition of the G19–C56 base pair (PDB ID: 4V4I). (D) T-box riboswitch recognition of the G19–C56 base pair (PDB ID: 4LCK). PRORP1 PPR domain, RNase P RNA, 23S rRNA and T-box riboswitch in green are shown as cartoons with interacting residues displayed as stick models. Amino acid side chains and tRNA D-loop (light blue) and TψC-loop (orange) nucleotides are shown with atom colors: oxygen (red), nitrogen (blue), phosphorus (orange) and sulfur (yellow). Non-tRNA interacting nucleotides of bacterial RNase P, 23S rRNA and T-box riboswitch RNAs are shown as thin stick models.