RT Journal Article SR Electronic T1 A nucleotidyltransferase toxin inhibits growth of Mycobacterium tuberculosis through inactivation of tRNA acceptor stems JF Science Advances JO Sci Adv FD American Association for the Advancement of Science SP eabb6651 DO 10.1126/sciadv.abb6651 VO 6 IS 31 A1 Cai, Yiming A1 Usher, Ben A1 Gutierrez, Claude A1 Tolcan, Anastasia A1 Mansour, Moise A1 Fineran, Peter C. A1 Condon, Ciarán A1 Neyrolles, Olivier A1 Genevaux, Pierre A1 Blower, Tim R. YR 2020 UL http://advances.sciencemag.org/content/6/31/eabb6651.abstract AB 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.