Aminoacyl-tRNA synthetases attach specific amino acids to cognate tRNAs. Prolyl-tRNA synthetases are known to mischarge tRNA Pro with the smaller amino acid alanine and with cysteine, which is the same size as proline. Quality control in proline codon translation is partly ensured by an editing domain (INS) present in most bacterial prolyl-tRNA synthetases that hydrolyzes smaller Ala-tRNA Pro Aminoacyl-tRNA synthetases (aaRSs) 3 play a pivotal role in the decoding of genetic information by catalyzing the esterification of cognate amino acids onto specific transfer RNAs (tRNAs) in a two-step reaction (1). The first step involves amino acid activation with ATP to form an aminoacyl-adenylate intermediate and concomitant pyrophosphate release. The second step involves aminoacyl transfer to the cognate tRNA via a transesterification reaction. Although aaRSs display high substrate specificity, they often misactivate structurally similar amino acids. If left unchecked, these mistakes lead to errors in protein synthesis, and accumulation of such errors can be deleterious to cells (2, 3).To ensure high fidelity in translation, aaRSs have evolved several proofreading mechanisms (4 -7). In the first type of proofreading, misactivated aminoacyl-adenylates are enzymatically hydrolyzed or selectively released from the active site followed by solvent hydrolysis, in a process termed "pretransfer" editing. Another mechanism of proofreading known as "posttransfer" editing is generally believed to function via the socalled "double-sieve" mechanism (8). The model predicted that, whereas one active site could not completely discriminate Ile and Val, two separate active sites with distinct strategies for recognition could significantly enhance fidelity. The aminoacylation active site of the aaRS would act as a "coarse" sieve for adenylate synthesis, activating the cognate amino acid but also allowing, to a lesser extent, activation of isosteric or smaller amino acids that could fit into the amino acid binding pocket. The second "fine" sieve would selectively bind misactivated amino acids for editing while excluding the original cognate amino acid. Thus, substrates synthesized in the first sieve would be further screened by the second sieve to enhance fidelity. Subsequently, biochemical and structural data validated this steric exclusion mechanism for valyl-tRNA synthetase and isoleucyl-tRNA synthetase (9, 10). Indeed, members of both classes of synthetases are now known to possess a second active site spatially separated from the ancient catalytic core to carry out post-transfer editing. Structures of class I isoleucyl-tRNA synthetase (10), leucyl-tRNA synthetase (11), and valyl-tRNA synthetase (12) reveal the highly conserved connective peptide 1 domain that functions in editing. Class II alanyl-tRNA synthetase (AlaRS) (13, 14), threonyl-tRNA synthetase (15), prolyltRNA synthetase (ProRS) (16,17), and phenylalanyl-tRNA synthetase (18, 19) also possess distinct domains for post-transfer editing. A recent study of AlaRS revealed an ...

Nuclear RNA exosomes catalyze a range of RNA processing and decay activities that are coordinated in part by cofactors, including Mpp6, Rrp47, and the Mtr4 RNA helicase. Mpp6 interacts with the nine-subunit exosome core, while Rrp47 stabilizes the exoribonuclease Rrp6 and recruits Mtr4, but it is less clear if these cofactors work together. Using biochemistry with Saccharomyces cerevisiae proteins, we show that Rrp47 and Mpp6 stimulate exosome-mediated RNA decay, albeit with unique dependencies on elements within the nuclear exosome. Mpp6-exosomes can recruit Mtr4, while Mpp6 and Rrp47 each contribute to Mtr4-dependent RNA decay, with maximal Mtr4-dependent decay observed with both cofactors. The 3.3 Å structure of a twelve-subunit nuclear Mpp6 exosome bound to RNA shows the central region of Mpp6 bound to the exosome core, positioning its Mtr4 recruitment domain next to Rrp6 and the exosome central channel. Genetic analysis reveals interactions that are largely consistent with our model.DOI: http://dx.doi.org/10.7554/eLife.29062.001

Background: Proofreading by aminoacyl-tRNA synthetases is a key translational quality control step. Results: The alanine specificity of the prolyl-tRNA synthetase editing domain can be modulated by mutations within the conserved hydrophobic binding pocket. Conclusion: Modulation of the editing domain specificity by mutation confirms a size exclusion-based mechanism. Significance: Distinct editing mechanisms are required to clear mischarged tRNA species and ensure accuracy of translation.

Errors in protein synthesis due to mispairing of amino acids with tRNAs jeopardize cell viability. Several checkpoints to prevent formation of Ala- and Cys-tRNAPro 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-tRNAPro in trans. In many species where ProRS lacks an INS domain, ProXp-ala, another single-domain INS-like protein, is responsible for editing Ala-tRNAPro. 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.