A nucleotidyltransferase toxin inhibits growth of Mycobacterium tuberculosis through inactivation of tRNA acceptor stems

Toxin-antitoxin systems are widespread stress-responsive elements, many of whose functions remain largely unknown. Here, we characterize the four DUF1814-family nucleotidyltransferase-like toxins (MenT_1-4) encoded by the human pathogen Mycobacterium tuberculosis. Toxin MenT₃ inhibited growth of M. tuberculosis when not antagonized by its cognate antitoxin, MenA₃. We solved the structures of toxins MenT₃ and MenT₄ to 1.6 and 1.2 Å resolution, respectively, and identified the biochemical activity and target of MenT₃. MenT₃ blocked in vitro protein expression and prevented tRNA charging in vivo. MenT₃ added pyrimidines (C or U) to the 3'-CCA acceptor stems of uncharged tRNAs and exhibited strong substrate specificity in vitro, preferentially targeting tRNA^Ser from among the 45 M. tuberculosis tRNAs. Our study identifies a previously unknown mechanism that expands the range of enzymatic activities used by bacterial toxins, uncovering a new way to block protein synthesis and potentially treat tuberculosis and other infections.