Abstract
Type II toxin–antitoxins systems are widespread in prokaryotic genomes. Typically, they comprise two proteins, a toxin, and an antitoxin, encoded by adjacent genes and forming a complex in which the enzymatic activity of the toxin is inhibited. Under stress conditions, the antitoxin is degraded liberating the active toxin. Though thousands of various toxin–antitoxins pairs have been predicted bioinformatically, only a handful has been thoroughly characterized. Here, we describe the AtaT2 toxin from a toxin–antitoxin system from Escherichia coli O157:H7. We show that AtaT2 is the first GNAT (Gcn5-related N-acetyltransferase) toxin that specifically targets charged glycyl tRNA. In vivo, the AtaT2 activity induces ribosome stalling at all four glycyl codons but does not evoke a stringent response. In vitro, AtaT2 acetylates the aminoacyl moiety of isoaccepting glycyl tRNAs, thus precluding their participation in translation. Our study broadens the known target specificity of GNAT toxins beyond the earlier described isoleucine and formyl methionine tRNAs, and suggest that various GNAT toxins may have evolved to specificaly target other if not all individual aminoacyl tRNAs.
Highlights
Prokaryotic toxin–antitoxin (TA) modules encode a proteinaceous toxin and a cognate antitoxin, which can be a protein or an RNA [1]
To test whether the toxicity of AtaT2 depends on its acetyltransferase activity, we expressed the wild-type AtaT2 alone or together with its cognate antitoxin AtaR2, and the catalytically-inactive AtaT2 mutant using E. coli BW25113 cells which lack an AtaRT2 module of their own
To test whether the two components of the AtaRT2 module form a complex, we tagged the AtaT2 toxin with a streptag, and the AtaR2 antitoxin with a hexahistidine tag and the dual construct was expressed in E. coli cells
Summary
Prokaryotic toxin–antitoxin (TA) modules encode a proteinaceous toxin and a cognate antitoxin, which can be a protein or an RNA [1]. TAs are classified based on the nature of the antitoxin and mechanism of toxin inactivation. Antitoxins of the type II TA modules are proteins that bind their cognate toxins and form inactive complexes [10]. The mechanisms that lead to toxin activation remain elusive and are subject of debate [11]. A less common class of type II toxins is comprised of proteins that are characterized by the Gcn5-related N-acetyltransferase (GNAT) fold [8,9]. The GNAT class of toxins is monophyletic and contains hundreds of predicted members found in sequenced bacterial genomes [13]. Toxicity of several others was verified in vivo, but the tar-
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