{ "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.", "abstract_shown": true, "affiliations": [ "Molecular Biophysics Unit, Indian Institute of Science, Bengaluru, Karnataka, India." ], "article_type": "Research Article", "arxiv": null, "author_total": 7, "authors": [ { "affs": [ 0 ], "name": "Kavyashree Nadig" }, { "affs": [ 0 ], "name": "Surekha Padmanaban" }, { "affs": [ 0 ], "name": "Salik Noor" }, { "affs": [ 0 ], "name": "Sankaran Sandhya" }, { "affs": [ 0 ], "name": "Parthasarathy Manikandan" }, { "affs": [ 0 ], "name": "Somnath Dutta" }, { "affs": [ 0 ], "name": "Mahavir Singh", "orcid": "0000-0001-7310-292X" } ], "bibtex": "https://research.draconitic.com/paper/f9swdxeeb7f90.bib", "cited_by": 0, "doi": "10.1371/journal.ppat.1014690", "fulltext_url": "https://journals.plos.org/plospathogens/article?id=10.1371/journal.ppat.1014690", "id": "f9swdxeeb7f90", "issns": [ "1553-7374", "1553-7366" ], "landing_url": "https://journals.plos.org/plospathogens/article?id=10.1371/journal.ppat.1014690", "language": "eng", "licence": "CC-BY", "licence_label": "CC BY", "licence_source": "article", "licence_url": "https://creativecommons.org/licenses/by/4.0/", "metadata_licence": null, "native_subjects": [ [ "openalex", "2303|Bacteriophages and microbial interactions" ], [ "plos", "/Biology and life sciences/Biochemistry/Nucleic acids/RNA/RNA structure" ], [ "plos", "/Biology and life sciences/Biochemistry/Nucleic acids/RNA/RNA structure/Pseudoknots" ], [ "plos", "/Biology and life sciences/Biochemistry/Proteins/Ribonucleoproteins" ], [ "plos", "/Biology and life sciences/Developmental biology/Microbial growth and development/Bacterial growth" ], [ "plos", "/Biology and life sciences/Microbiology/Medical microbiology/Microbial pathogens/Bacterial pathogens/Escherichia coli" ], [ "plos", "/Biology and life sciences/Microbiology/Microbial physiology/Microbial growth and development/Bacterial growth" ], [ "plos", "/Biology and life sciences/Molecular biology/Macromolecular structure analysis/RNA structure" ], [ "plos", "/Biology and life sciences/Molecular biology/Macromolecular structure analysis/RNA structure/Pseudoknots" ], [ "plos", "/Biology and life sciences/Organisms/Bacteria/Enterobacteriaceae/Escherichia/Escherichia coli" ], [ "plos", "/Biology and life sciences/Organisms/Bacteria/Gut bacteria/Escherichia/Escherichia coli" ], [ "plos", "/Biology and life sciences/Toxicology/Antitoxins" ], [ "plos", "/Biology and life sciences/Toxicology/Toxic agents/Toxins" ], [ "plos", "/Medicine and health sciences/Pathology and laboratory medicine/Pathogens/Microbial pathogens/Bacterial pathogens/Escherichia coli" ], [ "plos", "/Medicine and health sciences/Pathology and laboratory medicine/Toxicology/Antitoxins" ], [ "plos", "/Medicine and health sciences/Pathology and laboratory medicine/Toxicology/Toxic agents/Toxins" ], [ "plos", "/Research and analysis methods/Animal studies/Experimental organism systems/Model organisms/Escherichia coli" ], [ "plos", "/Research and analysis methods/Animal studies/Experimental organism systems/Prokaryotic models/Escherichia coli" ], [ "plos", "/Research and analysis methods/Microscopy/Electron microscopy/Electron cryo-microscopy" ], [ "plos", "/Research and analysis methods/Model organisms/Escherichia coli" ] ], "openalex": "W7220735207", "pdf_url": "https://journals.plos.org/plospathogens/article/file?id=10.1371/journal.ppat.1014690&type=printable", "pmcid": "PMC13682666", "pmid": "42837402", "published": "2026-10-06", "publisher": "Public Library of Science", "ris": "https://research.draconitic.com/paper/f9swdxeeb7f90.ris", "sources": [ { "first_seen": "2026-10-30T12:17:37Z", "native_id": "10.1371/journal.ppat.1014690", "source": "plos", "url": "https://journals.plos.org/plospathogens/article?id=10.1371/journal.ppat.1014690" }, { "first_seen": "2026-10-30T12:18:02Z", "native_id": "MED:42837402", "source": "europepmc", "url": "https://europepmc.org/article/MED/42837402" } ], "subjects": [ { "field": "biochemistry", "origin": "plos:molecular biology", "subfield": "molecular-biology" }, { "field": "biology", "origin": "plos:microbiology", "subfield": "microbiology" }, { "field": "medicine", "origin": "plos:toxicology", "subfield": "pharmacology-toxicology" } ], "title": "Cryo-EM reveals the quaternary architecture of TenpIN type III toxin-antitoxin RNP complex common to pathogenic bacteria", "title_text": "Cryo-EM reveals the quaternary architecture of TenpIN type III toxin-antitoxin RNP complex common to pathogenic bacteria", "type": "peer-reviewed", "updated_at": "2026-10-30T12:18:02Z", "url": "https://research.draconitic.com/paper/f9swdxeeb7f90", "venue": "PLOS Pathogens", "versions": [] }