Mechanism of translation inhibition by type II GNAT toxin AtaT2

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.

Comparative Transcriptome Analysis Reveals Mechanisms of Folate Accumulation in Maize Grains

“…Increasing the ability to acetylate ρ-ABAGlu enhances folate metabolism and demonstrates that NAT regulates folate levels by acetylating ρ-ABAGlu in animals [45], while whether NAT in plants is related to ρ-ABAGlu remains unknown. Aminoacyl-tRNA, which is formylated to form THF to participate in one-carbon metabolism [46], also is acetylated by GNAT toxins in Arabidopsis [47]. Here, genes belonging to the GNAT-transcription factor family were significantly enriched, while more evidence is required to validate if GNAT contributes to folate accumulation in maize.…”

Molecular basis of glycyl-tRNAGly acetylation by TacT from Salmonella Typhimurium

“…The AtaT2 encoded on the E. coli O157:H7 chromosome was recently suggested to be a Gly-tRNA Gly -specific toxin (Ovchinnikov et al, 2020), while AtaT from E. coli O157:H7 acetylates Gly-tRNA Gly , Trp-tRNA Trp , Phe-tRNA Phe , Tyr-tRNA Tyr , and Met-tRNAf Met in vivo and in vitro (Yashiro et al, 2020). In the report, ribosomal profiling in AtaT2-expressing E. coli cells showed that AtaT2 expression induces ribosome stalling on Gly codons in the A-site of the ribosome, and AtaT2 also acetylates Gly-tRNA Gly in vitro (Ovchinnikov et al, 2020). However, the mechanism of the specific acetylation of Gly-tRNA Gly by AtaT2 remained enigmatic.…”

“…During the past several years, a new type-II TA module in which the toxin belongs to the Gcn5-related N-acetyltransferase (GNAT) family was identified in various bacteria (Cheverton et al, 2016;Jur _ enas et al, 2017aJur _ enas et al, , 2017bMcVicker and Tang, 2016;Ovchinnikov et al, 2020;Qian et al, 2018;Rycroft et al, 2018;Wilcox et al, 2018;Yeo, 2018). The GNAT toxin family includes AtaT and AtaT2 from enterohemorrhagic Escherichia coli O157:H7 (Jur _ enas et al, 2017a; Ovchinnikov et al, 2020); ItaT from the E. coli HS strain (Wilcox et al, 2018); TacT, TacT2, and TacT3 from Salmonella Typhimurium and Salmonella Enteritidis (Cheverton et al, 2016;Rycroft et al, 2018); KacT from Klebsiella pneumoniae (Qian et al, 2018;Yeo, 2018); GmvT from Shigella sonnei (McVicker and Tang, 2016), and others. GNAT toxins acetylate the a-amino group of the aminoacyl moiety of aminoacyl-tRNAs (aa-tRNAs), using acetyl-coenzyme A (AcCoA) as the acetyl group donor, thus inhibiting cellular protein synthesis.…”

“…GNAT toxins acetylate the a-amino group of the aminoacyl moiety of aminoacyl-tRNAs (aa-tRNAs), using acetyl-coenzyme A (AcCoA) as the acetyl group donor, thus inhibiting cellular protein synthesis. The GNAT toxins from various bacteria reportedly target different aminoacyl-tRNA species and diverge in their specificities toward aminoacyl-tRNAs (Jur _ enas et al, 2017a;Ovchinnikov et al, 2020;Rycroft et al, 2018;Yashiro et al, 2020;Zhang et al, 2020). However, the molecular basis for the different substrate specificities of the GNAT family toxins has remained elusive.…”

“…Thus, the substrate selection by AtaT was suggested to be governed by the specific acceptor stem sequence of tRNA and the properties of the aminoacyl moieties of aminoacyl-tRNAs (Yashiro et al, 2020). On the other hand, AtaT2 was suggested to acetylate Gly-tRNA Gly specifically in vivo, through a ribosomeprofile analysis in AtaT2-induced cells; however, the mechanism of the specific acetylation of Gly-tRNA Gly by AtaT2 has remained elusive (Ovchinnikov et al, 2020).…”