Toxin-antitoxin (TA) systems

Toxin-antitoxin (TA) systems, including the well-studied mazEF operon, play key roles in bacterial 26 persistence, biofilm formation, programmed cell death, and stress responses. These physiological 27 manifestations stem from the endoribonuclease activity of the MazF toxin. Earlier studies on 28 Escherichia coli MazF determined that this enzyme exhibits cleavage specificity at precise recognition 29 sequences within single-stranded RNA. As a result, MazF family members were proposed to function by 30 targeting mRNAs, and these targets became the predominant focus for understanding how MazF affects 31 bacterial physiology. However, recent data indicate that MazF toxins can cleave specific sites within rRNAs 32 in concert with mRNAs. We identified the repertoire of RNAs cleaved by one of the nine Mycobacterium 33 tuberculosis toxins, 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 34 D-loop or within the tRNALys43 35 anticodon. By testing MazF-mt9-mediated cleavage of a series of altered tRNA- 36 based substrates, we demonstrated that this highly selective discrimination of RNA targets is achieved 37 through recognition of not only sequence but also structural determinants, a property not documented for 38 any other MazF toxin. Interestingly, the tRNase activity of MazF-mt9 mirrors basic features of eukaryotic 39 tRNases that also generate stable tRNA-derived fragments that can inhibit translation in response to 40 stress. Our data also suggests a role for tRNA distinct from its hallmark adapter function in translation, as 41 cleavage of tRNAs by MazF-mt9 leads to downregulation of cell growth.