A nucleotidyltransferase toxin inhibits growth of <i>Mycobacterium tuberculosis</i> 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 MenT 3 inhibited growth of M. tuberculosis when not antagonized by its cognate antitoxin, MenA 3 . We solved the structures of toxins MenT 3 and MenT 4 to 1.6 and 1.2 Å resolution, respectively, and identified the biochemical activity and target of MenT 3 . MenT 3 blocked in vitro protein expression and prevented tRNA charging in vivo. MenT 3 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.