Leucyl-tRNA synthetase (LeuRS) produces error free leucyl-tRNALeu by coordinating translocation of the 3′ end of (mis-)charged tRNAs from its synthetic site to a separate proof-reading site for editing. Here we report co-crystal structures of the Escherichia coli LeuRS-tRNALeu complex in the aminoacylation or editing conformations and show that translocation involves correlated rotations of four flexibly linked LeuRS domains. This pivots the tRNA to guide the charged tRNA 3′ end from the closed aminoacylation state to the editing site. The editing domain unexpectedly stabilizes the tRNA during aminoacylation while a large rotation of the leucine-specific domain positions the conserved KMSKS loop to bind the 3′ end of the tRNA, promoting catalysis. Our results give new insight into the structural dynamics of a molecular machine that is essential for accurate protein synthesis.

g Gram-negative bacteria cause approximately 70% of the infections in intensive care units. A growing number of bacterial isolates responsible for these infections are resistant to currently available antibiotics and to many in development. Most agents under development are modifications of existing drug classes, which only partially overcome existing resistance mechanisms. Therefore, new classes of Gram-negative antibacterials with truly novel modes of action are needed to circumvent these existing resistance mechanisms. We have previously identified a new a way to inhibit an aminoacyl-tRNA synthetase, leucyl-tRNA synthetase (LeuRS), in fungi via the oxaborole tRNA trapping (OBORT) mechanism. Herein, we show how we have modified the OBORT mechanism using a structure-guided approach to develop a new boron-based antibiotic class, the aminomethylbenzoxaboroles, which inhibit bacterial leucyl-tRNA synthetase and have activity against Gram-negative bacteria by largely evading the main efflux mechanisms in Escherichia coli and Pseudomonas aeruginosa. The lead analogue, AN3365, is active against Gram-negative bacteria, including Enterobacteriaceae bearing NDM-1 and KPC carbapenemases, as well as P. aeruginosa. This novel boronbased antibacterial, AN3365, has good mouse pharmacokinetics and was efficacious against E. coli and P. aeruginosa in murine thigh infection models, which suggest that this novel class of antibacterials has the potential to address this unmet medical need.

Influenza virus polymerase initiates the biosynthesis of its own mRNAs with capped 10-to 13-nucleotide fragments cleaved from cellular (pre-)mRNAs. Two activities are required for this cap-snatching activity: specific binding of the cap structure and an endonuclease activity. Recent work has shown that the cap-binding site is situated in the central part of the PB2 subunit and that the endonuclease activity is situated in the N-terminal domain of the PA subunit (PA-Nter). The influenza endonuclease is a member of the PD-(D/E)XK family of nucleases that use divalent metal ions for nucleic acid cleavage. Here we analyze the metal binding and endonuclease activities of eight PA-Nter single-point mutants. We show by calorimetry that the wild-type active site binds two Mn 2؉ ions and has a 500-fold higher affinity for manganese than for magnesium ions. The endonuclease activity of the isolated mutant domains are compared with the cap-dependent transcription activities of identical mutations in trimeric recombinant polymerases previously described by other groups. Mutations that inactivate the endonuclease activity in the isolated PA-Nter knock out the transcription but not replication activity in the recombinant polymerase. We confirm the importance of a number of active-site residues and identify some residues that may be involved in the positioning of the RNA substrate in the active site. Our results validate the use of the isolated endonuclease domain in a drug-design process for new anti-influenza virus compounds.

Epigenetic silencing of transposons by Piwi-interacting RNAs (piRNAs) constitutes an RNA-based genome defense mechanism. Piwi endonuclease action amplifies the piRNA pool by generating new piRNAs from target transcripts by a poorly understood mechanism. Here, we identified mouse Fkbp6 as a factor in this biogenesis pathway delivering piRNAs to the Piwi protein Miwi2. Mice lacking Fkbp6 derepress LINE1 (L1) retrotransposon and display reduced DNA methylation due to deficient nuclear accumulation of Miwi2. Like other cochaperones, Fkbp6 associates with the molecular chaperone Hsp90 via its tetratricopeptide repeat (TPR) domain. Inhibition of the ATP-dependent Hsp90 activity in an insect cell culture model results in the accumulation of short antisense RNAs in Piwi complexes. We identify these to be byproducts of piRNA amplification that accumulate only in nuage-localized Piwi proteins. We propose that the chaperone machinery normally ejects these inhibitory RNAs, allowing turnover of Piwi complexes for their continued participation in piRNA amplification.

The recent development and spread of extensively drug-resistant and totally drug-resistant resistant (TDR) strains of Mycobacterium tuberculosis highlight the need for new antitubercular drugs. Protein synthesis inhibitors have played an important role in the treatment of tuberculosis (TB) starting with the inclusion of streptomycin in the first combination therapies. Although parenteral aminoglycosides are a key component of therapy for multidrug-resistant TB, the oxazolidinone linezolid is the only orally available protein synthesis inhibitor that is effective against TB. Here, we show that small-molecule inhibitors of aminoacyl-tRNA synthetases (AARSs), which are known to be excellent antibacterial protein synthesis targets, are orally bioavailable and effective against M. tuberculosis in TB mouse infection models. We applied the oxaborole tRNA-trapping (OBORT) mechanism, which was first developed to target fungal cytoplasmic leucyl-tRNA synthetase (LeuRS), to M. tuberculosis LeuRS. X-ray crystallography was used to guide the design of LeuRS inhibitors that have good biochemical potency and excellent whole-cell activity against M. tuberculosis. Importantly, their good oral bioavailability translates into in vivo efficacy in both the acute and chronic mouse models of TB with potency comparable to that of the frontline drug isoniazid.

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Toxoplasma gondii is an important food and waterborne pathogen causing toxoplasmosis, a potentially severe disease in immunocompromised or congenitally infected humans. Available therapeutic agents are limited by suboptimal efficacy and frequent side effects that can lead to treatment discontinuation. Here we report that the benzoxaborole AN3661 had potent in vitro activity against T. gondii. Parasites selected to be resistant to AN3661 had mutations in TgCPSF3, which encodes a homologue of cleavage and polyadenylation specificity factor subunit 3 (CPSF‐73 or CPSF3), an endonuclease involved in mRNA processing in eukaryotes. Point mutations in TgCPSF3 introduced into wild‐type parasites using the CRISPR/Cas9 system recapitulated the resistance phenotype. Importantly, mice infected with T. gondii and treated orally with AN3661 did not develop any apparent illness, while untreated controls had lethal infections. Therefore, Tg CPSF3 is a promising novel target of T. gondii that provides an opportunity for the development of anti‐parasitic drugs.

The fidelity of protein synthesis depends on the capacity of aminoacyl-tRNA synthetases (AARSs) to couple only cognate amino acidtRNA pairs. If amino acid selectivity is compromised, fidelity can be ensured by an inherent AARS editing activity that hydrolyses mischarged tRNAs. Here, we show that the editing activity of Escherichia coli leucyl-tRNA synthetase (EcLeuRS) is not required to prevent incorrect isoleucine incorporation. Rather, as shown by kinetic, structural and in vivo approaches, the prime biological function of LeuRS editing is to prevent mis-incorporation of the non-standard amino acid norvaline. This conclusion follows from a reassessment of the discriminatory power of LeuRS against isoleucine and the demonstration that a LeuRS editing-deficient E. coli strain grows normally in high concentrations of isoleucine but not under oxygen deprivation conditions when norvaline accumulates to substantial levels. Thus, AARS-based translational quality control is a key feature for bacterial adaptive response to oxygen deprivation. The non-essential role for editing under normal bacterial growth has important implications for the development of resistance to antimicrobial agents targeting the LeuRS editing site.