Cryo-EM reveals the quaternary architecture of TenpIN type III toxin-antitoxin RNP complex common to pathogenic bacteria.

Abstract

Type III toxin-antitoxin (TA) systems are anti-phage defense systems in bacteria that comprises a protein toxin and a cognate non-coding RNA antitoxin. Under normal conditions, the antitoxin binds and inhibits the toxin. Upon phage infection, antitoxin is neutralized, releasing the active toxin, which cleaves the bacterial as well as phage RNA and halts viral replication. These systems are classified into tenpIN, cptIN, and toxIN families based on toxin sequence homology. While the ToxIN and CptIN RNP complexes have been structurally characterized, TenpIN systems remained poorly understood for their structure and assembly. Here, we report the functional and structural characterization of a TenpIN system found in several pathogenic bacteria. The antitoxin RNA, tenpI, contains two non-identical repeats, both capable of binding TenpN toxin, and rescuing toxin-induced growth inhibition in E. coli. Using single-particle cryo-electron microscopy and de novo modeling, we determined the structure of the TenpIN complex as a closed hetero-tetramer comprising two TenpN proteins and two tenpI RNAs. The RNA folds into a central pseudoknot flanked by single-stranded regions interacting with the toxin. Structural analysis revealed a close resemblance between TenpN and ToxN, while tenpI RNA shares structural features with cptI RNA, thereby expanding our understanding of the structural diversity of Type III TA systems.