Substrate recognition and cleavage-site selection by a single-subunit protein-only RNase P

Abstract

RNase P is the enzyme that removes 5' extensions from tRNA precursors. With its diversity of enzyme forms-either protein- or RNA-based, ranging from single polypeptides to multi-subunit ribonucleoproteins-the RNase P enzyme family represents a unique model system to compare the evolution of enzymatic mechanisms. Here we present a comprehensive study of substrate recognition and cleavage-site selection by the nuclear single-subunit proteinaceous RNase P PRORP3 from Arabidopsis thaliana. Compared to bacterial RNase P, the best-characterized RNA-based enzyme form, PRORP3 requires a larger part of intact tRNA structure, but little to no determinants at the cleavage site or interactions with the 5' or 3' extensions of the tRNA. The cleavage site depends on the combined dimensions of acceptor stem and T domain, but also requires the leader to be single-stranded. Overall, the single-subunit PRORP appears mechanistically more similar to the complex nuclear ribonucleoprotein enzymes than to the simpler bacterial RNase P. Mechanistic similarity or dissimilarity among different forms of RNase P thus apparently do not necessarily reflect molecular composition or evolutionary relationship.

Figure 1.

Structure of Thermus thermophilus pre-tRNA^Gly, derived deletion variants and minimal substrates. (A) Classical cloverleaf representation of pre-tRNA^Gly. The structural domains are color-coded: magenta, aminoacyl acceptor stem; blue, D domain; red, anticodon domain; gold, variable domain; green, TΨC domain. The positions of selected nucleotides are numbered according to convention (70). The canonical RNase P cleavage site is between nucleotides −1 and 1. (B) Predicted secondary structures of pre-tRNA^Gly variants without anticodon (Ac) or D domain, or composed of the aminoacyl acceptor stem (Aa) and TΨC domain (T) only, some with a bulge (_b) of variable length inserted; the sequence of all 5′ leaders (not shown) is identical to wild-type pre-tRNA^Gly shown in (A).

Figure 2.

Tertiary-structure position of conserved nucleotides whose identity was altered. Two-dimensional representations of the (L-shaped) tertiary structures (domains color-coded as in Figure 1) with tertiary interactions indicated by broken lines; solid gray lines indicate phosphodiester bonds of adjacent nucleotides that are displayed distant in the two-dimensional representation of the tertiary structure. (A) Structure of pre-tRNA^Gly and position of conserved nucleotides in the TΨC or D loop that were altered in the substrate variants; base substitutions with nucleotide number indicated. (B) Predicted structure of the minimal substrate Aa_b9T and position of conserved nucleotides in the TΨC loop that were altered in the substrate variants.

Figure 3.

Nucleotide-specifying residues of plant-PRORP PPR motifs. Conjectural nucleotide-specifying residues of the five PPR motifs found in PRORPs of Chloroplastida/Viridiplantae. From the structure of Arabidopsis thaliana PRORP1 (30) the nucleotide-specifying residues (NSR) 1, 4 and 34 (,; numbering according to the Pfam PPR model) of the PPR motifs of the three A. thaliana PRORPs were derived and a sequence logo was generated from the alignment of 175 PRORP sequences.

Figure 4.

Cleavage-site selection by PRORP3. Variants of pre-tRNA^Gly and the minimal-substrate Aa_b9T with varying length and structure of the aminoacyl acceptor stem and/or T domain, or pre-tRNA^Gly variants with base-paired nucleotides −1 and 73 were subjected to cleavage by PRORP3, and the cleavage site determined by mapping the length of the released 5′ leader. Processing assays with the different substrates were incubated with different concentrations of PRORP3 (for pre-tRNA^Gly wild-type, Aa_−2bp, Aa_+2bp and wild-type Aa_b9T: 200 nM; for Aa_b9T Aa_+2bp, T_+2bp, T_+2bp Aa_−2bp and T_4loop: 1 μM; for pre-tRNA^Gly Aa_+4bp, Aa_+m3GC and Aa_+m3AU: 500 nM; for pre-tRNA^His, pre-tRNA^Gly G₋₁–C₇₃, U₋₁–A₇₃ and Aa_+3AU: 200 nM) or with 10 nM Bacillus subtilis RNase P (pre-tRNA^His) until sufficient product had formed. RNAs were separated by 12% (B, D and F) or 15% (G and I) denaturing PAGE (only the part showing the 5′-cleavage products is shown). Alkaline hydrolysis ladders were generated from wild-type pre-tRNA^Gly (due to 2′,3′-cyclic-phosphate ends their migration is slightly offset relative to the RNase P cleavage products with 3′-hydroxyl ends). (A) Pre-tRNA^Gly variants with an acceptor stem extended or shortened by inserting or deleting 2 bp; the other tRNA domains (not shown) are identical to the wild-type (see Figure 1A). The indicated reference positions 1 and −1 are for the purpose of this study defined as the seventh and eighth nucleotide in the 5′ strand of the aminoacyl acceptor stem counting (in 3′-to-5′ direction) from the base of the stem. Arrows of different size indicate major and minor cleavage sites. (B) Cleavage-site determination of the variants shown in (A). (C) Acceptor stem and T domain variants of the minimal substrate Aa_b9T. (D) Cleavage-site determination of the variants shown in (C). (E) Pre-tRNA^Gly variants with an acceptor stem extended by 4, by a mismatch and 3 G–C, or by a mismatch and 3 A–U bp. (F) Cleavage-site determination of the variants shown in (E). (G) Cleavage of E. coli pre-tRNA^His by B. subtilis RNase P and PRORP3. (H) Pre-tRNA^Gly variants with base-paired nucleotides −1 and 73, or with an acceptor stem extended by 3 A–U bp. (I) Cleavage-site determination of the variants shown in (H).