Structural basis of tRNAPro acceptor stem recognition by a bacterial trans-editing domain.

High fidelity tRNA aminoacylation by aminoacyl-tRNA synthetases is essential for cell viability. ProXp-ala is a trans-editing protein that is present in all three domains of life and is responsible for hydrolyzing mischarged Ala-tRNAPro and preventing mistranslation of proline codons. Previous studies have shown that, like bacterial prolyl-tRNA synthetase, Caulobacter crescentus ProXp-ala recognizes the unique C1:G72 terminal base pair of the tRNAPro acceptor stem, helping to ensure deacylation of Ala-tRNAPro but not Ala-tRNAAla. The structural basis for C1:G72 recognition by ProXp-ala is still unknown and was investigated here. NMR spectroscopy, binding, and activity assays revealed two conserved residues, K50 and R80, that likely interact with the first base pair, stabilizing the initial protein-RNA encounter complex. Modeling studies are consistent with direct interaction between R80 and the major groove of G72. A third key contact between A76 of tRNAPro and K45 of ProXp-ala was essential for binding and accommodating the CCA-3' end in the active site. We also demonstrated the essential role that the 2'OH of A76 plays in catalysis. Eukaryotic ProXp-ala proteins recognize the same acceptor stem positions as their bacterial counterparts, albeit with different nucleotide base identities. ProXp-ala is encoded in some human pathogens; thus, these results have the potential to inform new antibiotic drug design.

Antibiotic that inhibits trans-translation blocks binding of EF-Tu to tmRNA but not to tRNA.

IMPORTANCE: Elongation factor thermo-unstable (EF-Tu) is a universally conserved translation factor that mediates productive interactions between tRNAs and the ribosome. In bacteria, EF-Tu also delivers transfer-messenger RNA (tmRNA)-SmpB to the ribosome during trans-translation. We report the first small molecule, KKL-55, that specifically inhibits EF-Tu activity in trans-translation without affecting its activity in normal translation. KKL-55 has broad-spectrum antibiotic activity, suggesting that compounds targeted to the tmRNA-binding interface of EF-Tu could be developed into new antibiotics to treat drug-resistant infections.

Trans-editing by aminoacyl-tRNA synthetase-like editing domains.

Aminoacyl-tRNA synthetases (aaRS) are ubiquitous enzymes responsible for aminoacyl-tRNA (aa-tRNA) synthesis. Correctly formed aa-tRNAs are necessary for proper decoding of mRNA and accurate protein synthesis. tRNAs possess specific nucleobases that promote selective recognition by cognate aaRSs. Selecting the cognate amino acid can be more challenging because all amino acids share the same peptide backbone and several are isosteric or have similar side chains. Thus, aaRSs can misactivate non-cognate amino acids and produce mischarged aa-tRNAs. If left uncorrected, mischarged aa-tRNAs deliver their non-cognate amino acid to the ribosome resulting in misincorporation into the nascent polypeptide chain. This changes the primary protein sequence and potentially causes misfolding or formation of non-functional proteins that impair cell survival. A variety of proofreading or editing pathways exist to prevent and correct mistakes in aa-tRNA formation. Editing may occur before the amino acid transfer step of aminoacylation via hydrolysis of the aminoacyl-adenylate. Alternatively, post-transfer editing, which occurs after the mischarged aa-tRNA is formed, may be carried out via a distinct editing site on the aaRS where the mischarged aa-tRNA is deacylated. In recent years, it has become clear that most organisms also encode factors that lack aminoacylation activity but resemble aaRS editing domains and function to clear mischarged aa-tRNAs in trans. This review focuses on these trans-editing factors, which are encoded in all three domains of life and function together with editing domains present within aaRSs to ensure that the accuracy of protein synthesis is sufficient for cell survival.