The Mycobacterium tuberculosis genome contains an unusually high number of toxin-antitoxin modules, some of which have been suggested to play a role in the establishment and maintenance of latent tuberculosis. Nine of these toxin-antitoxin loci belong to the mazEF family, encoding the intracellular toxin MazF and its antitoxin inhibitor MazE. Nearly every MazF ortholog recognizes a unique three-or five-base RNA sequence and cleaves mRNA. As a result, these toxins selectively target a subset of the transcriptome for degradation and are known as "mRNA interferases." Here we demonstrate that a MazF family member from M. tuberculosis, MazF-mt6, has an additional role-inhibiting translation through targeted cleavage of 23S rRNA in the evolutionarily conserved helix/loop 70. We first determined that MazF-mt6 cleaves mRNA at 5′ UU↓CCU 3′ sequences. We then discovered that MazF-mt6 also cleaves M. tuberculosis 23S rRNA at a single UUCCU in the ribosomal A site that contacts tRNA and ribosome recycling factor. To gain further mechanistic insight, we demonstrated that MazFmt6-mediated cleavage of rRNA can inhibit protein synthesis in the absence of mRNA cleavage. Finally, consistent with the position of 23S rRNA cleavage, MazF-mt6 destabilized 50S-30S ribosomal subunit association. Collectively, these results show that MazF toxins do not universally act as mRNA interferases, because MazF-mt6 inhibits protein synthesis by cleaving 23S rRNA in the ribosome active center.T oxin-antitoxin (TA) systems have the potential to control Mycobacterium tuberculosis growth rate and persistence. One of the best characterized TA modules is mazEF in Escherichia coli (1-7), an autoregulated operon that encodes the intracellular toxin MazF and its antitoxin inhibitor MazE. Under unstressed conditions, the MazE protein forms a stable complex with MazF to neutralize its toxicity (1). During times of stress, however, proteases degrade MazE and allow the relatively stable MazF toxin to disrupt protein synthesis (1, 2), which can induce a state of reversible dormancy (3). Expression of MazF triggers this quasi-dormant state, during which cells stop dividing but are able to transcribe mRNA and synthesize proteins (4). The striking similarities between this state of quasi-dormancy and the slowgrowing or nonreplicating state of M. tuberculosis during latent tuberculosis (TB) have led to the suggestion that TA modules are involved with persistence and dormancy in M. tuberculosis (8).The genome of M. tuberculosis has >80 putative TA pairs (9), a remarkably large number relative to most other prokaryotes. Although TA modules are ubiquitous in bacteria and archaea, few prokaryotes have more than 15 loci (10). Although the physiological role of the large repertoire of TA loci in M. tuberculosis is largely unknown, some clues have emerged. First, there is an inverse correlation between the number of chromosomal TA loci and growth rate (10). Second, more than 60 TA toxins are conserved between five members of the M. tuberculosis complex but not in 13 oth...

Toxin-antitoxin (TA) systems play key roles in bacterial persistence, biofilm formation and stress responses. The MazF toxin from the Escherichia coli mazEF TA system is a sequence- and single-strand-specific endoribonuclease, and many studies have led to the proposal that MazF family members exclusively target mRNA. However, recent data indicate some MazF toxins can cleave specific sites within rRNA in concert with mRNA. In this report, we identified the repertoire of RNAs cleaved by Mycobacterium tuberculosis toxin MazF-mt9 using an RNA-seq-based approach. This analysis revealed that two tRNAs were the principal targets of MazF-mt9, and each was cleaved at a single site in either the tRNAPro14 D-loop or within the tRNALys43 anticodon. This highly selective target discrimination occurs through recognition of not only sequence but also structural determinants. Thus, MazF-mt9 represents the only MazF family member known to target tRNA and to require RNA structure for recognition and cleavage. Interestingly, the tRNase activity of MazF-mt9 mirrors basic features of eukaryotic tRNases that also generate stable tRNA-derived fragments that can inhibit translation in response to stress. Our data also suggest a role for tRNA distinct from its canonical adapter function in translation, as cleavage of tRNAs by MazF-mt9 downregulates bacterial growth.