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 (MenT1-4) 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 tRNASer 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.