MqsR is a noncanonical microbial RNase toxin that is inhibited by antitoxin MqsA via steric blockage of substrate binding.

The MqsRA toxin-antitoxin system is a component of the Escherichia coli stress response. Free MqsR, a ribonuclease, cleaves mRNAs containing a 5'-GC-3' sequence causing a global shutdown of translation and the cell to enter a state of dormancy. Despite a general understanding of MqsR function, the molecular mechanism(s) by which MqsR binds and cleaves RNA and how one or more of these activities is inhibited by its cognate antitoxin MqsA is still poorly understood. Here, we used NMR spectroscopy coupled with mRNA cleavage assays to identify the molecular mechanism of MqsR substrate recognition and the MqsR residues that are essential for its catalytic activity. We show that MqsR preferentially binds substrates that contain purines in the -2 and -1 position relative to the MqsR consensus cleavage sequence and that two residues of MqsR, Tyr81, and Lys56 are strictly required for mRNA cleavage. We also show that MqsA inhibits MqsR activity by sterically blocking mRNA substrates from binding while leaving the active site fully accessible to mononucleotides. Together, these data identify the residues of MqsR that mediate RNA cleavage and reveal a novel mechanism that regulates MqsR substrate specificity.