2. Academic Validation
  3. Sequence-specific trapping of EF-Tu/glycyl-tRNA complex on the ribosome by bottromycin

Sequence-specific trapping of EF-Tu/glycyl-tRNA complex on the ribosome by bottromycin

  • bioRxiv. 2025 Aug 20:2025.08.17.670399. doi: 10.1101/2025.08.17.670399.
Dmitrii Y Travin 1 2,  Ritwika S Basu 3,  Madhura N Paranjpe 4,  Dorota Klepacki 1 2,  Anna I Zhurakovskaya 1 2 3 4 5 6 7,  Nora Vázquez-Laslop 1 2,  Alexander S Mankin 1 2,  Yury S Polikanov 1 2 4,  Matthieu G Gagnon 3 5 6 7
Affiliations

Affiliations

  • 1 Department of Pharmaceutical Sciences, University of Illinois at Chicago, Chicago, IL 60607, USA.
  • 2 Center for Biomolecular Sciences, University of Illinois at Chicago, Chicago, IL 60607, USA.
  • 3 Department of Microbiology and Immunology, University of Texas Medical Branch, Galveston, TX 77555, USA.
  • 4 Department of Biological Sciences, University of Illinois at Chicago, Chicago, IL 60607, USA.
  • 5 Department of Biochemistry and Molecular Biology, University of Texas Medical Branch, Galveston, TX 77555, USA.
  • 6 Sealy Center for Structural Biology and Molecular Biophysics, University of Texas Medical Branch, Galveston, TX 77555, USA.
  • 7 Institute for Human Infections and Immunity, University of Texas Medical Branch, Galveston, Texas 77555, USA.
PMID: 40894528 DOI: 10.1101/2025.08.17.670399
Abstract

The development of Antibiotics with novel mechanisms of action is essential to address the growing threat of antimicrobial resistance. Protein synthesis-inhibiting Antibiotic bottromycin (BOT), a ribosomally synthesized and posttranslationally modified peptide (RiPP), has long been known for its potent activity against Gram-positive bacteria but was largely neglected due in part to the lack of understanding of its mechanism of action. Here we uncover the unprecedented mode translation inhibition strategy employed by BOT. Using biochemical, microbiological, genetic, and structural approaches, we show that BOT acts by selectively trapping elongation factor-Tu (EF-Tu) in complex with glycyl-tRNA on the ribosome. BOT binds at the interface between EF-Tu and the CCA-end of Gly-tRNA, stabilizing the EF-Tu/Gly-tRNA complex in a pre-accommodated A/T-state on the ribosome, and specifically arresting translation at glycine codons. This mode of action is mechanistically distinct from that of Other EF-Tu-targeting Antibiotics, which act in a tRNA-agnostic fashion. Point mutations in EF-Tu confer high-level resistance to BOT, confirming EF-Tu as the direct and essential target of the drug. Our findings establish BOT as a founding member of a new class of Antibiotics that stall the ribosome at defined mRNA sites by trapping a specific elongation factor-tRNA complex.

Keywords

EF-Tu; Elfamycins; antibiotic; bottromycin; context-specificity; cryo-EM; protein synthesis inhibitor; translation.

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