Role of Antitoxin RNA Pseudoknot in Regulating Toxin Activity and Toxin-antitoxin RNP Complex Assembly

Toxin-antitoxin (TA) systems are bacterial defense systems that confer survival advantages under stress. TA systems comprise a toxin and an antitoxin gene, usually present as operon on chromosomes or on plasmids in bacteria. In type III ToxIN TA systems toxin gene encodes a protein toxin (ToxN) which is a sequence-specific endoribonuclease and antitoxin gene encodes an RNA antitoxin (ToxI) that neutralizes toxin by forming a closed-cyclic TA RNP complex. In TA assemblies, antitoxin RNA adopts a complex tertiary structure comprising of a central conserved pseudoknot flanked by toxin-binding 5' and 3' single-stranded regions. In this study, we have shown that a closed, cyclic assembly of ToxIN RNP complex is required for the complete ToxN inhibition in E. coli. We have probed tertiary contacts within the antitoxin pseudoknot that are essential for toxin inhibition in E. coli. Furthermore, we investigated the impact of several ToxI mutants on antitoxin RNA stability, structure, and TA complex assembly using in vitro biophysical and biochemical experiments. We have shown that ToxI mutants adopt structures different from the functional ToxI repeat. In altered conformations, ToxI mutants were able to bind the toxin but were unable to assemble into closed assemblies, resulting in incomplete inhibition of the toxin. Our findings showed that subtle nucleotide changes in the pseudoknot can disrupt antitoxin-mediated toxin neutralization, emphasizing its role in TA complex assembly.