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Toxin-antitoxin (TA) loci are prevalent in bacterial genomes. They are suggested to play a central role in dormancy and persister states. Under normal growth conditions, TA toxins are neutralized by their cognate antitoxins, and under stress conditions, toxins are freed and inhibit essential cellular processes using a variety of mechanisms. Here we characterize ataR-ataT, a novel TA system, from enterohemorrhagic Escherichia coli. We show that the toxin AtaT is a GNAT family enzyme that transfers an acetyl group from acetyl coenzyme A to the amine group of the methionyl aminoacyl moiety of initiator tRNA. AtaT specifically modifies Met-tRNA fMet , but no other aminoacyl-tRNAs, including the elongator Met-tRNA Met . We demonstrate that once acetylated, AcMet-tRNA fMet fails to interact with initiation factor-2 (IF2), resulting in disruption of the translation initiation complex. This work reveals a new mechanism of translation inhibition and confirms Met-tRNA fMet as a prime target to efficiently block cell growth.

The ataRT operon from enteropathogenic Escherichia coli encodes a toxin–antitoxin (TA) module with a recently discovered novel toxin activity. This new type II TA module targets translation initiation for cell‐growth arrest. Virtually nothing is known regarding the molecular mechanisms of neutralization, toxin catalytic action or translation autoregulation. Here, the production, biochemical analysis and crystallization of the intrinsically disordered antitoxin AtaR, the toxin AtaT, the AtaR–AtaT complex and the complex of AtaR–AtaT with a double‐stranded DNA fragment of the operator region of the promoter are reported. Because they contain large regions that are intrinsically disordered, TA antitoxins are notoriously difficult to crystallize. AtaR forms a homodimer in solution and crystallizes in space group P6122, with unit‐cell parameters a = b = 56.3, c = 160.8 Å. The crystals are likely to contain an AtaR monomer in the asymmetric unit and diffracted to 3.8 Å resolution. The Y144F catalytic mutant of AtaT (AtaTY144F) bound to the cofactor acetyl coenzyme A (AcCoA) and the C‐terminal neutralization domain of AtaR (AtaR44–86) were also crystallized. The crystals of the AtaTY144F–AcCoA complex diffracted to 2.5 Å resolution and the crystals of AtaR44–86 diffracted to 2.2 Å resolution. Analysis of these structures should reveal the full scope of the neutralization of the toxin AtaT by AtaR. The crystals belonged to space groups P6522 and P3121, with unit‐cell parameters a = b = 58.1, c = 216.7 Å and a = b = 87.6, c = 125.5 Å, respectively. The AtaR–AtaT–DNA complex contains a 22 bp DNA duplex that was optimized to obtain high‐resolution data based on the sequence of two inverted repeats detected in the operator region. It crystallizes in space group C2221, with unit‐cell parameters a = 75.6, b = 87.9, c = 190.5 Å. These crystals diffracted to 3.5 Å resolution. [ABSTRACT FROM AUTHOR]

The article reports on the analysis of the capacity of phosphorylation to deactivate the translation of elongation factor Tu (EF-Tu) in Escherichia coli bacteria in a conformational dynamics. Topics include the association between bacterial protein synthesis and metabolic rate, the structural and biophysical evidences of the role of phosphorylation in decelerating protein synthesis through decoupling of nucleotide binding, and the role of direct modifications of EF-Tu switch region.