Mechanism of translation inhibition by type II GNAT toxin AtaT2.

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.

Structural Basis of Leader Peptide Recognition in Lasso Peptide Biosynthesis Pathway.

Lasso peptides are a class of ribosomally synthesized and post-translationally modified peptides (RiPPs) with a unique 3D-interlocked structure, in which an N-terminal macrolactam ring is threaded by a linear C-terminal part. The unique structure of lasso peptides is introduced into ribosomally translated precursor peptides by lasso peptide synthetase encompassing proteins B and C or B1, B2, and C when the B enzyme is split into two distinct proteins. The B1 protein recognizes the leader sequence of the precursor peptide, and then the B2 protein cleaves it. The C protein catalyzes the formation of the macrolactam ring. However, the detailed mechanism of lasso peptide maturation has remained elusive, due to the lack of structural information about the responsible proteins. Here we report the crystal structure of the B1 protein from the thermophilic actinobacteria, Thermobifida fusca (TfuB1), complexed with the leader peptide (TfuA-Leader), which revealed the detailed mechanism of leader peptide recognition. The structure of TfuB1 consists of an N-terminal ß-sheet and three C-terminal helices. The leader peptide is docked on one edge of the N-terminal ß-sheet of TfuB1, as an additional ß strand. Three conserved amino acid residues of the leader peptide (TfuA Tyr-17, Pro-14, and Leu-12) fit well on the hydrophobic cleft between the ß-sheet and adjacent helices. Biochemical analysis demonstrated that these conserved residues are essential for affinity between TfuB1 and the TfuA-Leader. Furthermore, we found that TfuB1 and the leader peptide jointly form a hydrophobic patch on the ß-sheet, which includes the highly conserved TfuA Phe-6 and TfuB1 Tyr33. Homology modeling and mutational analysis of the B1 protein from a firmicute, Bacillus pseudomycoides (PsmB1), revealed that the hydrophobic patch is conserved in a wide range of species and involved in the cleavage activity of the B2 protein, indicating it forms the interaction surface for the B2 protein or the core part of the precursor peptide.

Escherichia coli ItaT is a type II toxin that inhibits translation by acetylating isoleucyl-tRNAIle.

Prokaryotic toxin-antitoxin (TA) modules are highly abundant and are involved in stress response and drug tolerance. The most common type II TA modules consist of two interacting proteins. The type II toxins are diverse enzymes targeting various essential intracellular targets. The antitoxin binds to cognate toxin and inhibits its function. Recently, TA modules whose toxins are GNAT-family acetyltransferases were described. For two such systems, the target of acetylation was shown to be aminoacyl-tRNA: the TacT toxin targets aminoacylated elongator tRNAs, while AtaT targets the amino acid moiety of initiating tRNAMet. We show that the itaRT gene pair from Escherichia coli encodes a TA module with acetyltransferase toxin ItaT that specifically and exclusively acetylates Ile-tRNAIle thereby blocking translation and inhibiting cell growth. ItaT forms a tight complex with the ItaR antitoxin, which represses the transcription of itaRT operon. A comprehensive bioinformatics survey of GNAT acetyltransferases reveals that enzymes encoded by validated or putative TA modules are common and form a distinct branch of the GNAT family tree. We speculate that further functional analysis of such TA modules will result in identification of enzymes capable of specifically targeting many, perhaps all, aminoacyl tRNAs.