Mechanism of aminoacyl-tRNA acetylation by an aminoacyl-tRNA acetyltransferase AtaT from enterohemorrhagic E. coli

Abstract

Toxin-antitoxin systems in bacteria contribute to stress adaptation, dormancy, and persistence. AtaT, a type-II toxin in enterohemorrhagic E. coli, reportedly acetylates the α-amino group of the aminoacyl-moiety of initiator Met-tRNAfMet, thus inhibiting translation initiation. Here, we show that AtaT has a broader specificity for aminoacyl-tRNAs than initially claimed. AtaT efficiently acetylates Gly-tRNAGly, Trp-tRNATrp, Tyr-tRNATyr and Phe-tRNAPhe isoacceptors, in addition to Met-tRNAfMet, and inhibits global translation. AtaT interacts with the acceptor stem of tRNAfMet, and the consecutive G-C pairs in the bottom-half of the acceptor stem are required for acetylation. Consistently, tRNAGly, tRNATrp, tRNATyr and tRNAPhe also possess consecutive G-C base-pairs in the bottom halves of their acceptor stems. Furthermore, misaminoacylated valyl-tRNAfMet and isoleucyl-tRNAfMet are not acetylated by AtaT. Therefore, the substrate selection by AtaT is governed by the specific acceptor stem sequence and the properties of the aminoacyl-moiety of aminoacyl-tRNAs.

RelA-SpoT Homolog toxins pyrophosphorylate the CCA end of tRNA to inhibit protein synthesis

“…In order to understand the biological roles of toxSAS TAs, it will be essential to study these effectors and the antitoxins neutralizing them in native bacterial and viral hosts. Finally, exhaustive tRNA specificity studies similar to those undertaken for GNAT AtaT TA toxins (Yashiro et al, 2020) are necessary in order to establish the specific cellular tRNA species targeted by translation-targeting toxSAS toxins.…”

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

“…The fractions were then treated with stable isotopic acetic anhydride-D6 ((CD 3 CO) 2 O, where D is deuterium) to chemically acetylate the remaining aminoacyl-tRNAs that were not acetylated by the action of TacT in cells. The RNAs were then digested with RNase I, and the hydrolysates were analyzed by liquid chromatographymass spectrometry (LC-MS) (Yashiro et al, 2020;Zhang et al, 2020) (Figure 1A).…”

“…To elucidate the molecular mechanism of the selective acetylation of Gly-tRNA Gly by TacT, we co-crystallized the catalytic mutant TacT with the Y140F mutation (Cheverton et al, 2016), (Yashiro et al, 2020). (B) LC-MS analysis of RNase-I-digested fragments of acetyl-aminoacyl-tRNAs after 30 min of induced TacT expression (pBAD-TacT) and the negative control (pBAD).…”

“…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.…”

“…AtaT also recognizes the properties of the aminoacyl moieties of aminoacyl-tRNAs. 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).…”