Cryo-EM reveals the quaternary architecture of TenpIN type III toxin-antitoxin RNP complex common to pathogenic bacteria
- 1Molecular Biophysics Unit, Indian Institute of Science, Bengaluru, Karnataka, India.
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 antitoxin 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. Author summary: Bacteria have evolved sophisticated mechanisms to survive stressful conditions and defend themselves against invading bacteriophages. Toxin-antitoxin (TA) systems are one such defence mechanism. They consist of a toxin that can inhibit bacterial growth and an antitoxin that keeps the toxin inactive under normal conditions. In type III TA systems, the antitoxin is an RNA molecule that directly binds and neutralizes the protein toxin. Although several type III TA systems have been studied, the tenpIN family remains poorly understood in terms of its structure and assembly. Recent bioinformatic analyses have revealed that tenpIN systems are much more widespread than previously recognized, including in important antibiotic-resistant bacterial pathogens, highlighting the importance of understanding their molecular mechanisms. Here, we report the functional characterization, purification, and quaternary structure of a TenpIN ribonucleoprotein complex that is conserved across several pathogenic bacteria. The structure revealed that the TenpIN complex contains two toxin proteins (TenpN) and two antitoxin RNA (tenpI repeats) molecules. We have also identified and experimentally validated putative active-site residues of TenpN. These findings expand our understanding of the structural and functional diversity of type III TA systems and provide a foundation for exploring their roles in bacterial defence, biotechnology, and antimicrobial research.
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BibTeX
@article{nadig2026cryoem,
title = {{Cryo-EM reveals the quaternary architecture of TenpIN type III toxin-antitoxin RNP complex common to pathogenic bacteria}},
author = {Kavyashree Nadig and Surekha Padmanaban and Salik Noor and Sankaran Sandhya and Parthasarathy Manikandan and Somnath Dutta and Mahavir Singh},
journal = {PLOS Pathogens},
year = {2026},
month = oct,
doi = {10.1371/journal.ppat.1014690},
url = {https://journals.plos.org/plospathogens/article?id=10.1371/journal.ppat.1014690},
note = {CC BY},
}
RIS
TY - JOUR
TI - Cryo-EM reveals the quaternary architecture of TenpIN type III toxin-antitoxin RNP complex common to pathogenic bacteria
AU - Kavyashree Nadig
AU - Surekha Padmanaban
AU - Salik Noor
AU - Sankaran Sandhya
AU - Parthasarathy Manikandan
AU - Somnath Dutta
AU - Mahavir Singh
PY - 2026
DA - 2026/10/06
JO - PLOS Pathogens
DO - 10.1371/journal.ppat.1014690
UR - https://journals.plos.org/plospathogens/article?id=10.1371/journal.ppat.1014690
AB - 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 antitoxin 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. Author summary: Bacteria have evolved sophisticated mechanisms to survive stressful conditions and defend themselves against invading bacteriophages. Toxin-antitoxin (TA) systems are one such defence mechanism. They consist of a toxin that can inhibit bacterial growth and an antitoxin that keeps the toxin inactive under normal conditions. In type III TA systems, the antitoxin is an RNA molecule that directly binds and neutralizes the protein toxin. Although several type III TA systems have been studied, the tenpIN family remains poorly understood in terms of its structure and assembly. Recent bioinformatic analyses have revealed that tenpIN systems are much more widespread than previously recognized, including in important antibiotic-resistant bacterial pathogens, highlighting the importance of understanding their molecular mechanisms. Here, we report the functional characterization, purification, and quaternary structure of a TenpIN ribonucleoprotein complex that is conserved across several pathogenic bacteria. The structure revealed that the TenpIN complex contains two toxin proteins (TenpN) and two antitoxin RNA (tenpI repeats) molecules. We have also identified and experimentally validated putative active-site residues of TenpN. These findings expand our understanding of the structural and functional diversity of type III TA systems and provide a foundation for exploring their roles in bacterial defence, biotechnology, and antimicrobial research.
SN - 1553-7374
SN - 1553-7366
ER -