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. 2013 May 17;288(20):14391-14399.
doi: 10.1074/jbc.M113.467795. Epub 2013 Apr 5.

Exclusive use of trans-editing domains prevents proline mistranslation

Oscar Vargas-Rodriguez  1 , Karin Musier-Forsyth  2
Affiliations

Exclusive use of trans-editing domains prevents proline mistranslation

Oscar Vargas-Rodriguez et al. J Biol Chem. .

Abstract

Aminoacyl-tRNA synthetases (ARSs) catalyze the attachment of specific amino acids to cognate tRNAs. Although the accuracy of this process is critical for overall translational fidelity, similar sizes of many amino acids provide a challenge to ARSs. For example, prolyl-tRNA synthetases (ProRSs) mischarge alanine and cysteine onto tRNA(Pro). Many bacterial ProRSs possess an alanine-specific proofreading domain (INS) but lack the capability to edit Cys-tRNA(Pro). Instead, Cys-tRNA(Pro) is cleared by a single-domain homolog of INS, the trans-editing YbaK protein. A global bioinformatics analysis revealed that there are six types of "INS-like" proteins. In addition to INS and YbaK, four additional single-domain homologs are widely distributed throughout bacteria: ProXp-ala (formerly named PrdX), ProXp-x (annotated as ProX), ProXp-y (annotated as YeaK), and ProXp-z (annotated as PA2301). The last three are domains of unknown function. Whereas many bacteria encode a ProRS containing an INS domain in addition to YbaK, many other combinations of INS-like proteins exist throughout the bacterial kingdom. Here, we focus on Caulobacter crescentus, which encodes a ProRS with a truncated INS domain that lacks catalytic activity, as well as YbaK and ProXp-ala. We show that C. crescentus ProRS can readily form Cys- and Ala-tRNA(Pro), and deacylation studies confirmed that these species are cleared by C. crescentus YbaK and ProXp-ala, respectively. Substrate specificity of C. crescentus ProXp-ala is determined, in part, by elements in the acceptor stem of tRNA(Pro) and further ensured through collaboration with elongation factor Tu. These results highlight the diversity of approaches used to prevent proline mistranslation and reveal a novel triple-sieve mechanism of editing that relies exclusively on trans-editing factors.

Keywords: Aminoacyl tRNA Synthesis; Aminoacyl tRNA Synthetase; Post-transfer Editing; Proofreading; Transfer RNA (tRNA); Translation; Translation Elongation Factors.

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Figures

FIGURE 1.
FIGURE 1.
Phylogeny of INS-like proteins in bacteria. The color of the branches defines the identity of each family (blue, INS; red, YbaK; green, ProXp-ala; black, ProXp-x; pink, ProXp-y; brown, ProXp-z), and the new names of the family members are shown with the previous name in parentheses. The leaf colors define the aa-tRNA specificity of each family: red, Cys-tRNA; blue, Ala-tRNA, and gray, unknown specificity. Branches displaying a bootstrap support >80% are indicated by a gray circle.
FIGURE 2.
FIGURE 2.
Misacylation and pre-transfer editing activities of C. crescentus ProRS. A, formation of mischarged Ala-tRNAPro by 0.5 μm WT C. crescentus (Cc) ProRS (▾), 0.5 μm E. coli (Ec) ProRS (●), and 0.5 μm E. coli K279A ProRS (○). Reactions were carried out at 37 °C with 5 μm E. coli tRNAPro. B, pre-transfer editing time course showing formation of AMP in the presence of proline (●), alanine (▾), and cysteine (■). Reaction conditions were as described under “Experimental Procedures.” Error bars, S.D. of triplicate determinations.
FIGURE 3.
FIGURE 3.
Post-transfer editing activities of C. crescentus YbaK, ProXp-ala, and ProRS. A, Cys-tRNAPro (0.4 μm) deacylation by 0.4 μm C. crescentus (Cc) YbaK (▴), 5 μm C. crescentus ProXp-ala (●), and 5 μm C. crescentus ProRS (▵). B, Ala-tRNAPro (0.75 μm) deacylation by 5 μm C. crescentus ProRS (■), 1 μm C. crescentus ProXp-ala (●), and 3 μm E. coli ProRS (○). Error bars, S.D. of triplicate determinations.
FIGURE 4.
FIGURE 4.
tRNA specificity of C. crescentus ProXp-ala. A, hydrolysis of E. coli Ala-tRNAs (∼0.75 μm) by 1 μm C. crescentus ProXp-ala. Substrates tested were WT Ala-tRNAPro (●), G1:C72/U70 Ala-tRNAPro (▾), C70U Ala-tRNAPro (○), and WT Ala-tRNAAla (▵). Error bars, S.D. of triplicate determinations. B, E. coli (Ec) tRNAPro and tRNAAla acceptor stem sequences with arrows pointing to mutations made in the context of full-length E. coli tRNAPro.
FIGURE 5.
FIGURE 5.
Effect of EF-Tu on ProXp-ala activity. A, alanine mischarging of E. coli tRNAPro (5 μm) by C. crescentus ProRS (1 μm). Reactions were performed in the presence (▿) and absence (♦) of 10 μm E. coli EF-Tu. Aminoacylation was in the presence of 1 μm C. crescentus ProXp-ala only (□) and C. crescentus ProXp-ala (1 μm) with E. coli EF-Tu (10 μm) (▴). A no enzyme control was also performed (●). B, alanine aminoacylation of E. coli tRNAAla (5 μm) by 50 nm E. coli AlaRS. Reactions were performed in the presence (♦) and absence of 10 μm E. coli EF-Tu (▿). Aminoacylation was in the presence of 1 μm C. crescentus ProXp-ala only (□) and C. crescentus ProXp-ala (1 μm) with E. coli EF-Tu (10 μm) (▴). A reaction with no enzyme was also performed (●). Error bars, S.D. of triplicate determinations.
FIGURE 6.
FIGURE 6.
Proposed triple-sieve editing mechanism in C. crescentus. C. crescentus ProRS lacks a full-length INS domain and readily misactivates alanine and cysteine. Two trans-editing domains, ProXp-ala and YbaK, function to deacylate Ala-tRNAPro and Cys-tRNAPro, respectively. The structures of R. palustris ProRS (27), C. crescentus ProXp-ala (27) and H. influenzae YbaK (28) are shown.
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