Distinct tRNA recognition strategies used by a homologous family of editing domains prevent mistranslation

Abstract

Errors in protein synthesis due to mispairing of amino acids with tRNAs jeopardize cell viability. Several checkpoints to prevent formation of Ala- and Cys-tRNA(Pro) have been described, including the Ala-specific editing domain (INS) of most bacterial prolyl-tRNA synthetases (ProRSs) and an autonomous single-domain INS homolog, YbaK, which clears Cys-tRNA(Pro) in trans. In many species where ProRS lacks an INS domain, ProXp-ala, another single-domain INS-like protein, is responsible for editing Ala-tRNA(Pro). Although the amino acid specificity of these editing domains has been established, the role of tRNA sequence elements in substrate selection has not been investigated in detail. Critical recognition elements for aminoacylation by bacterial ProRS include acceptor stem elements G72/A73 and anticodon bases G35/G36. Here, we show that ProXp-ala and INS require these same acceptor stem and anticodon elements, respectively, whereas YbaK lacks inherent tRNA specificity. Thus, these three related domains use divergent approaches to recognize tRNAs and prevent mistranslation. Whereas some editing domains have borrowed aspects of tRNA recognition from the parent aminoacyl-tRNA synthetase, relaxed tRNA specificity leading to semi-promiscuous editing may offer advantages to cells.

Figure 1.

Domain architecture of bacterial ProRSs, YbaK and ProXp-ala and deacylation activities of E. coli ProRS and C. crescentus ProXp-ala. (A) Three distinct architectures of bacterial ProRSs as represented by E. coli (Ec), C. crescentus (Cc) and T. thermophilus (Tt) ProRSs, with conserved motifs 1, 2 and 3 (M1–M3, black) and ABD (green). The C-terminal extension found in some bacterial ProRSs is shown in yellow. The editing domain (INS) of Ec ProRS and the truncated INS of Cc ProRS are shown in blue (11). INS-like single-domain proteins Ec YbaK and Cc ProXp-ala are shown in red and blue, respectively. Dotted lines indicate a gap. The column on the right indicates the known post-transfer editing activity of the corresponding enzyme. Ala and Cys indicate Ala-tRNA and Cys-tRNA deacylation, respectively. N/A indicates no deacylation activity. (B) Deacylation of Ala-[³²P]tRNA^Pro (filled circle) and Ala-[³²P]tRNA^Ala (filled triangle) by WT E. coli ProRS. Inset, deacylation of the same aminoacyl-tRNAs by C. crescentus ProXp-ala.

Figure 2.

Full-length tRNAs and E. coli tRNA^Pro acceptor stem variants tested as substrates for editing by E. coli ProRS and INS domain homologs. WT E. coli tRNA^Pro/UGG is shown (top left) with the acceptor stem region that is varied in this study (boxed). The acceptor stem variants of E. coli tRNA^Pro are also shown on the top with the altered sequences in bold-faced larger font. Microhelix^Pro (top right) is derived from the acceptor stem of E. coli tRNA^Pro and contain a stable UUCG tetraloop. Sequences of E. coli tRNA^Ala/UGC, human tRNA^Pro/UGG and E. coli tRNA^Cys/GCA also used in this study are shown at the bottom. The nucleotides previously identified as critical for aminoacylation of these tRNAs by the corresponding ARSs are circled (16–18,37,38). In the case of E. coli tRNA^Cys, the dotted circles indicate a critical tertiary core contact between G15:G48.

Figure 3.

Escherichia coli tRNA^Pro anticodon variants tested as substrates for deacylation by E. coli ProRS. Single substitutions of anticodon bases of E. coli tRNA^Pro selected for this study are shown on the left. Deacylation of WT Ala-tRNA^Pro anticodon variants by WT E. coli ProRS (right). The tRNA variants used here lack C1, which does not negatively affect aminoacylation (16) or deacylation (present study) by E. coli ProRS.

Figure 4.

Chimeric E. coli tRNA^Ala variants tested for hydrolysis by E. coli ProRS. (A) E. coli tRNA^Ala and anticodon domain variants studied here. Full-length tRNA^Ala variants containing Pro-specific anticodon bases UGG or GGG, or the entire Pro-specific anticodon loop (AC-Loop) or anticodon stem-loop (AC-SL) were tested. Changes relative to WT E. coli tRNA^Ala are shown in larger bold font. (B) Deacylation of [¹⁴C]-Ala-tRNA^Ala anticodon variants by WT E. coli ProRS (0.3 μM in Buffer B). Deacylation of G1:C72/U70 Ala-tRNA^Pro (tRNA^Pro) and WT Ala-tRNA^Ala are also shown.

Figure 5.

Divergent approaches for tRNA^Pro recognition by homologous INS-like editing domains. Bacterial ProRS recognizes acceptor stem elements G72/A73 and anticodon bases G35/G36 for efficient aminoacylation of tRNA^Pro. The cis-editing domain of ProRS (INS) depends on interactions of the ABD (green) with the anticodon bases of tRNA^Pro for hydrolysis of Ala-tRNA (indicated by dotted line). The trans-editing protein ProXp-ala relies only on the acceptor stem elements for hydrolysis. The YbaK trans-editing domain lacks tRNA recognition capability, but instead, interacts with ProRS to achieve tRNA specificity. The structures shown are Enterococcus faecalis ProRS (11), E. coli YbaK (47) and C. crescentus ProXp-ala (11).