Natural Homolog of tRNA Synthetase Editing Domain Rescues Conditional Lethality Caused by Mistranslation

Abstract

AlaXp is a widely distributed (from bacteria to humans) genome-encoded homolog of the editing domain of alanyltRNA synthetases. Editing repairs the confusion of serine and glycine for alanine through clearance of mischarged (with Ser or Gly) tRNA Ala . Because genome-encoded fragments of editing domains of other synthetases are scarce, the AlaXp redundancy of the editing domain of alanyl-tRNA synthetase is thought to reflect an unusual sensitivity of cells to mistranslation at codons for Ala. Indeed, a small defect in the editing activity of alanyltRNA synthetase is causally linked to neurodegeneration in the mouse. Although limited earlier studies demonstrated that AlaXp deacylated mischarged tRNA Ala in vitro, the significance of this activity in vivo has not been clear. Here we describe a bacterial system specifically designed to investigate activity of AlaXp in vivo. Serine toxicity, experienced by a strain harboring an editing-defective alanyl-tRNA synthetase, was rescued by an AlaXp-encoding transgene. Rescue was dependent on amino acid residues in AlaXp that are needed for its in vitro catalytic activity. Thus, the editing activity per se of AlaXp was essential for suppressing mistranslation. The results support the idea that the unique widespread distribution of AlaXp arises from the singular difficulties, for translation, poised by alanine.The editing activities of tRNA synthetases provide a major safeguard against mistranslation, the insertion of the wrong amino acid at a specific codon (1-4). This insertion arises from small amounts of tRNA mischarging, a phenomenon that is intrinsic to the active sites of many of the synthetases. This mischarging results from the inherent inability of the enzyme binding pockets to discriminate rigorously between closely similar amino acid side chains, especially to achieve the level of discrimination needed for the high accuracy of the genetic code. Examples include the synthesis of Val-tRNAIle by isoleucyl-tRNA synthetase (3, 5), Thr-tRNAVal by valyl-tRNA synthetase (6, 7), Val-tRNA Leu by leucyl-tRNA synthetase (8 -10), Ala-tRNA Pro by prolyl-tRNA synthetase (11, 12), Ser-tRNA Thr by threonyl-tRNA synthetase (13), Ile-tRNA Phe by phenylalanyl-tRNA synthetase (4), and Ser-or Gly-tRNA Ala by alanyltRNA synthetase (AlaRS) 2 (14, 15). Normally, the mischarged tRNAs are cleared by a distinct editing activity, specific to each synthetase. This activity is able to distinguish the correct from the incorrect amino acid fused to its cognate tRNA. In addition to the editing activity that is part of the synthetase and encoded as a separate domain (16 -18), there are a few instances of freestanding editing domain homologs encoded by various genomes (12, 19 -22). The most widespread (through all three kingdoms of life) of these is AlaXp, a small protein that is a homolog of the editing domain of AlaRS and has been shown to edit Ser-tRNA Ala and Gly-tRNA Ala in vitro (21-24). Not clear, however, is whether AlaXp plays a role in vivo in guarding against mistranslation.The importan...

Substrate-mediated Fidelity Mechanism Ensures Accurate Decoding of Proline Codons

“…Although AlaRSs edit both Gly-and Ser-tRNA Ala , freestanding AlaXps co-evolved as a second checkpoint to ensure editing of Ser-tRNA Ala , which poses a greater challenge than glycine (38,63). Although even mild editing defects have been linked to serious pathology in the mouse (2), the presence of ϳ10% mismade protein is not detrimental to E. coli (64).…”

Homologous trans -editing factors with broad tRNA specificity prevent mistranslation caused by serine/threonine misactivation

“…Cellular degradation and apoptosis caused by a mutation in the editing domain of ValRS have been reported in murine cells (16). In addition, editing defects in bacteria often result in slower growth rates, delayed growth, or even death (17)(18)(19)(20)(21)(22).…”

Yeast mitochondrial threonyl-tRNA synthetase recognizes tRNA isoacceptors by distinct mechanisms and promotes CUN codon reassignment

“…To maintain fidelity during protein synthesis, an aaRS thus needs to select the correct amino acid and tRNA substrates from a large pool of structurally similar molecules in the cell. The active-site groove of the aaRS plays a role of the first selection sieve that prevents activation of most noncognate amino acids (3,4), and a cis-editing site (5) or free standing editing domain (6)(7)(8) further proofreads misactivated amino acids. In contrast, the selection of tRNA substrates depends on the ability of aaRSs to establish interactions with a unique set of identity elements present in a given tRNA (9,10).…”