Synthesis of proteins containing errors (mistranslation) is prevented by aminoacyl transfer RNA synthetases through their accurate aminoacylation of cognate tRNAs and their ability to correct occasional errors of aminoacylation by editing reactions. A principal source of mistranslation comes from mistaking glycine or serine for alanine, which can lead to serious cell and animal pathologies, including neurodegeneration. A single specific G.U base pair (G3.U70) marks a tRNA for aminoacylation by alanyl-tRNA synthetase. Mistranslation occurs when glycine or serine is joined to the G3.U70-containing tRNAs, and is prevented by the editing activity that clears the mischarged amino acid. Previously it was assumed that the specificity for recognition of tRNA(Ala) for editing was provided by the same structural determinants as used for aminoacylation. Here we show that the editing site of alanyl-tRNA synthetase, as an artificial recombinant fragment, targets mischarged tRNA(Ala) using a structural motif unrelated to that for aminoacylation so that, remarkably, two motifs (one for aminoacylation and one for editing) in the same enzyme independently can provide determinants for tRNA(Ala) recognition. The structural motif for editing is also found naturally in genome-encoded protein fragments that are widely distributed in evolution. These also recognize mischarged tRNA(Ala). Thus, through evolution, three different complexes with the same tRNA can guard against mistaking glycine or serine for alanine.

The rules of the genetic code are established by aminoacylations of transfer RNAs by aminoacyl tRNA synthetases. New codon assignments, and the introduction of new kinds of amino acids, are blocked by vigorous tRNA-dependent editing reactions occurring at hydrolytic sites embedded within specialized domains in the synthetases. For some synthetases, these domains were present at the time of the last common ancestor and were fixed in evolution through all three of the kingdoms of life. Significantly, a well characterized domain for editing found in bacterial and eukaryotic threonyl-and all alanyl-tRNA synthetases is missing from archaebacterial threonine enzymes. Here we show that the archaebacterial Methanosarcina mazei ThrRS efficiently misactivates serine, but does not fuse serine to tRNA. Consistent with this observation, the enzyme cleared serine that was linked to threonine-specific tRNAs. M. mazei and most other archaebacterial ThrRSs have a domain, N2A, fused to the N terminus and not found in bacterial or eukaryotic orthologs. Mutations at conserved residues in this domain led to an inability to clear threonine-specific tRNA mischarged with serine. Thus, these results demonstrate a domain for editing that is distinct from all others, is restricted to just one branch of the tree of life, and was most likely added to archaebacterial ThrRSs after the eukaryote͞ archaebacteria split.A minoacyl tRNA synthetases are divided into two classes of 10 enzymes each, based on the active-site architecture shared by all members of the same class (1-4). This structural classification is based on the design of the ATP-binding and amino acid activation domain, which also interacts with the 3Ј end of tRNA. For both class I and class II enzymes, the class-defining domain is considered the ancient, historical tRNA synthetase and appeared at or before the time of the last common ancestor. Surrounding and permeating these central domains are insertions and N-or C-terminal fusions of additional motifs, many of which were added later in evolution. These motifs provide tRNA-binding determinants and, in some cases, functions extraneous to aminoacylation, such as transcriptional and translational control (5, 6), RNA splicing (7, 8), and cytokine signaling (9-13).In addition to these motifs, many synthetases have acquired a second active site that is designed to clear errors of aminoacylation (14 -19). Two widely distributed designs for these active sites for editing have been described, one for class I enzymes (17, 20 -24) and another for class II enzymes (25-27). In many instances, such as class I isoleucyl-, leucyl-, and valyl-tRNA synthetases, the domain for editing is conserved through evolution and is believed to have been present at the time of the split of the tree of life into three branches (28). In contrast, the universally conserved (in all three kingdoms) domain for editing in the class II alanyl-tRNA synthetase, although present in bacterial and eukaryote threonyl-tRNA synthetases, is missing in archaebacterial threony...

The toxicity of mistranslation of serine for alanine appears to be universal, and is prevented in part by the editing activities of alanyl-tRNA synthetases (AlaRSs), which remove serine from mischarged tRNA Ala . The problem of serine mistranslation is so acute that free-standing, genome-encoded fragments of the editing domain of AlaRSs are found throughout evolution. These AlaXps are thought to provide functional redundancy of editing. Indeed, archaeal versions rescue the conditional lethality of bacterial cells harboring an editing-inactive AlaRS. In mammals, AlaXps are encoded by a gene that fuses coding sequences of a homolog of the HSP90 cochaperone p23 (p23 H ) to those of AlaXp, to create p23 H AlaXp. Not known is whether this fusion protein, or various potential splice variants, are expressed as editing-proficient proteins in mammalian cells. Here we show that both p23 H AlaXp and AlaXp alternative splice variants can be detected as proteins in mammalian cells. The variant that ablated p23 H and encoded just AlaXp was active in vitro. In contrast, neither the p23 H AlaXp fusion protein, nor the mixture of free p23 H with AlaXp, was active. Further experiments in a mammalian cell-based system showed that RNAi-directed suppression of sequences encoding AlaXp led to a serine-sensitive increase in the accumulation of misfolded proteins. The results demonstrate the dependence of mammalian cell homeostasis on AlaXp, and implicate p23 H as a cis - and trans -acting regulator of its activity.

Alanyl-tRNA synthetase efficiently aminoacylates tRNA Ala and an RNA minihelix that comprises just one domain of the two-domain L-shaped tRNA structure. It also clears mischarged tRNA Ala using a specialized domain in its C-terminal half. In contrast to full-length tRNA Ala , minihelix Ala was robustly mischarged and could not be edited. Addition in trans of the missing anticodon-containing domain did not activate editing of mischarged minihelix Ala . To understand these differences between minihelix Ala and tRNA Ala , several chimeric full tRNAs were constructed. These had the acceptor stem of a non-cognate tRNA replaced with the stem of tRNA Ala . The chimeric tRNAs collectively introduced multiple sequence changes in all parts but the acceptor stem. However, although the acceptor stem in isolation (as the minihelix) lacked determinants for editing, alanyl-tRNA synthetase effectively cleared a mischarged amino acid from each chimeric tRNA. Thus, a covalently continuous two-domain structure per se, not sequence, is a major determinant for clearance of errors of aminoacylation by alanyl-tRNA synthetase. Because errors of aminoacylation are known to be deleterious to cell growth, structure-specific determinants constitute a powerful selective pressure to retain the format of the two-domain L-shaped tRNA.