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Cited by 11RNA and protein synthesis mechanismsRNA modifications and cancerBacterial Genetics and Biotechnology

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The translation inhibitors kasugamycin, edeine and GE81112 target distinct steps during 30S initiation complex formation

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Safdari, Haaris A., Morici, Martino, Sanchez-Castro, Ana, Dallapè, Andrea, Paternoga, Helge, Giuliodori, Anna Maria, Fabbretti, Attilio, Milón, Pohl, Wilson, Daniel N.·2025·Nature Communications
DOI 10.1038/s41467-025-57731-8PMC PMC11903750

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Abstract

During bacterial translation initiation, the 30S ribosomal subunit, initiation factors, and initiator tRNA define the reading frame of the mRNA. This process is inhibited by kasugamycin, edeine and GE81112, however, their mechanisms of action have not been fully elucidated. Here we present cryo-electron microscopy structures of 30S initiation intermediate complexes formed in the presence of kasugamycin, edeine and GE81112 at resolutions of 2.0-2.9 Å. The structures reveal that all three antibiotics bind within the E-site of the 30S and preclude 30S initiation complex formation. While kasugamycin and edeine affect early steps of 30S pre-initiation complex formation, GE81112 stalls pre-initiation complex formation at a further step by allowing start codon recognition, but impeding IF3 departure. Collectively, our work highlights how chemically distinct compounds binding at a conserved site on the 30S can interfere with translation initiation in a unique manner.

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  1. Milon, P., Konevega, A. L., Gualerzi, C. O. & Rodnina, M. V. Kinetic checkpoint at a late step in translation initiation. Mol. Cell 30, 712–720 (2008). · DOI
  2. Milon, P. & Rodnina, M. V. Kinetic control of translation initiation in bacteria. Crit. Rev. Biochem Mol. Biol. 47, 334–348 (2012). · DOI
  3. Gualerzi, C. O. & Pon, C. L. Initiation of mRNA translation in bacteria: structural and dynamic aspects. Cell Mol. Life Sci. 72, 4341–4367 (2015). · DOI
  4. Gualerzi, C. O., Fabbretti, A., Brandi, L., Milon, P. & Pon, C. L. Role of the Initiation Factors in mRNA Start Site Selection and fMet-tRNA Recruitment by Bacterial Ribosomes. Isr. J. Chem. 50, 80–94 (2010). · DOI
  5. Milon, P., Maracci, C., Filonava, L., Gualerzi, C. O. & Rodnina, M. V. Real-time assembly landscape of bacterial 30S translation initiation complex. Nat. Struct. Mol. Biol. 19, 609–615 (2012). · DOI
  6. Hussain, T., Llacer, J. L., Wimberly, B. T., Kieft, J. S. & Ramakrishnan, V. Large-Scale Movements of IF3 and tRNA during Bacterial Translation Initiation. Cell 167, 133–144 e13 (2016). · DOI
  7. Kaledhonkar, S. et al. Late steps in bacterial translation initiation visualized using time-resolved cryo-EM. Nature 570, 400–404 (2019). · DOI
  8. Nakamoto, J. A. et al. The dynamic cycle of bacterial translation initiation factor IF3. Nucleic Acids Res 49, 6958–6970 (2021). · DOI
  9. Goyal, A., Belardinelli, R., Maracci, C., Milon, P. & Rodnina, M. V. Directional transition from initiation to elongation in bacterial translation. Nucleic Acids Res 43, 10700–10712 (2015). · DOI
  10. Wilson, D. N. The A-Z of bacterial translation inhibitors. Crit. Rev. Biochem Mol. Biol. 44, 393–433 (2009). · DOI · Paper page
  11. Brandi, L. et al. Novel tetrapeptide inhibitors of bacterial protein synthesis produced by a Streptomyces sp. Biochemistry 45, 3692–3702 (2006). · DOI
  12. Giuliodori, A. M., Spurio, R., Milon, P. & Fabbretti, A. Antibiotics Targeting the 30S Ribosomal Subunit: A Lesson from Nature to Find and Develop New Drugs. Curr. Top. Med Chem. 18, 2080–2096 (2018). · DOI
  13. Umezawa, H., Hamada, M., Suhara, Y., Hashimoto, T. & Ikekawa, T. Kasugamycin, a new antibiotic. Antimicrobial Agents Chemother. 5, 753–757 (1965).
  14. Okuyama, A., Machiyama, N., Kinoshita, T. & Tanaka, N. Inhibition by kasugamycin of initiation complex formation on 30S ribosomes. Biochem. Biophys. Res. Commun. 43, 196–169 (1971). · DOI
  15. Poldermans, B., Goosen, N. & Van Knippenberg, P. H. Studies on the function of two adjacent N6,N6-dimethyladenosines near the 3’ end of 16 S ribosomal RNA of Escherichia coli. I. The effect of kasugamycin on initiation of protein synthesis. J. Biol. Chem. 254, 9085–9089 (1979). · DOI
  16. Schluenzen, F. et al. The antibiotic kasugamycin mimics mRNA nucleotides to destabilize tRNA binding and inhibit canonical translation initiation. Nat. Struct. Mol. Biol. 13, 871–878 (2006). · DOI
  17. Schuwirth, B. S. et al. Structural analysis of kasugamycin inhibition of translation. Nat. Struct. Mol. Biol. 13, 879–886 (2006). · DOI
  18. Paternoga, H. et al. Structural conservation of antibiotic interaction with ribosomes. Nat. Struct. Mol. Biol. 30, 1380–1392 (2023). · DOI · Paper page
  19. Okuyama, A., Tanaka, N. & Komai, T. The binding of kasugamycin to the Escherichia coli ribosomes. J. Antibiot. 28, 903–905 (1975). · DOI
  20. Woodcock, J., Moazed, D., Cannon, M., Davies, J. & Noller, H. F. Interaction of antibiotics with A- and P-site-specific bases in 16S ribosomal RNA. EMBO J. 10, 3099–3103 (1991). · DOI · Paper page
  21. Dinos, G. et al. Dissecting the ribosomal inhibition mechanisms of edeine and pactamycin: the universally conserved residues G693 and C795 regulate P-site tRNA binding. Mol. Cell 13, 113–124 (2004). · DOI
  22. Polikanov, Y. S., Aleksashin, N. A., Beckert, B. & Wilson, D. N. The Mechanisms of Action of Ribosome-Targeting Peptide Antibiotics. Front Mol. Biosci. 5, 48 (2018). · DOI
  23. Pioletti, M. et al. Crystal structures of complexes of the small ribosomal subunit with tetracycline, edeine and IF3. EMBO J. 20, 1829–1839 (2001). · DOI · Paper page
  24. Garreau de Loubresse, N. et al. Structural basis for the inhibition of the eukaryotic ribosome. Nature 513, 517–522 (2014). · DOI
  25. Brandi, L. et al. Specific, efficient, and selective inhibition of prokaryotic translation initiation by a novel peptide antibiotic. Proc. Natl Acad. Sci. USA 103, 39–44 (2006). · DOI
  26. Fabbretti, A. et al. Inhibition of translation initiation complex formation by GE81112 unravels a 16S rRNA structural switch involved in P-site decoding. Proc. Natl Acad. Sci. USA 113, E2286–E2295 (2016). · DOI
  27. Lopez-Alonso, J. P. et al. Structure of a 30S pre-initiation complex stalled by GE81112 reveals structural parallels in bacterial and eukaryotic protein synthesis initiation pathways. Nucleic Acids Res 45, 2179–2187 (2017).
  28. Vila-Sanjurjo, A., Squires, C. L. & Dahlberg, A. E. Isolation of kasugamycin resistant mutants in the 16 S ribosomal RNA of Escherichia coli. J. Mol. Biol. 293, 1–8 (1999). · DOI
  29. Jurjens, G. et al. Total Synthesis and Structural Revision of the Antibiotic Tetrapeptide GE81112A. Angew. Chem. Int Ed. Engl. 57, 12157–12161 (2018). · DOI
  30. Schuler, S. M. M. et al. Full Profiling of GE81112A, an Underexplored Tetrapeptide Antibiotic with Activity against Gram-Negative Pathogens. Microbiol Spectr. 11, e0224722 (2023). · DOI
  31. Schedlbauer, A. et al. A binding site for the antibiotic GE81112 in the ribosomal mRNA channel. bioRxiv https://doi.org/10.1101/2024.09.26.614503 (2024). · DOI
  32. Antoun, A., Pavlov, M. Y., Lovmar, M. & Ehrenberg, M. How initiation factors tune the rate of initiation of protein synthesis in bacteria. EMBO J. 25, 2539–2550 (2006). · DOI
  33. Zhang, Y. et al. The context of the ribosome binding site in mRNAs defines specificity of action of kasugamycin, an inhibitor of translation initiation. Proc Natl Acad Sci USA 119, e2118553119 (2022). · DOI
  34. Milon, P. et al. Transient kinetics, fluorescence, and FRET in studies of initiation of translation in bacteria. Methods Enzymol. 430, 1–30 (2007). · DOI
  35. Pestka, S. & Nirenberg, M. Regulatory mechanisms and protein synthesis. X. Codon recognition on 30 S ribosomes. J. Mol. Biol. 21, 145–171 (1966). · DOI
  36. Moazed, D., Van, S. B. J., Douthwaite, S. & Noller, H. F. Interconversion of active and inactive 30 S ribosomal subunits is accompanied by a conformational change in the decoding region of 16 S rRNA. J. Mol. Biol. 191, 483–493 (1986). · DOI
  37. Jahagirdar, D. et al. Alternative conformations and motions adopted by 30S ribosomal subunits visualized by cryo-electron microscopy. RNA 26, 2017–2030 (2020). · DOI
  38. Chulluncuy, R., Espiche, C., Nakamoto, J. A., Fabbretti, A. & Milon, P. Conformational Response of 30S-bound IF3 to A-Site Binders Streptomycin and Kanamycin. Antibiotics (Basel) 5, 38 (2016). · DOI
  39. Barrenechea, V., Vargas-Reyes, M., Quiliano, M. & Milon, P. A Complementary Mechanism of Bacterial mRNA Translation Inhibition by Tetracyclines. Front Microbiol 12, 682682 (2021). · DOI
  40. Wishnia, A., Boussert, A., Graffe, M., Dessen, P. H. & Grunberg-Manago, M. Kinetics of the reversible association of ribosomal subunits: stopped-flow studies of the rate law and of the effect of Mg2+. J. Mol. Biol. 93, 499–15 (1975). · DOI
  41. Kimanius, D., Dong, L., Sharov, G., Nakane, T. & Scheres, S. H. W. New tools for automated cryo-EM single-particle analysis in RELION-4.0. Biochem J. 478, 4169–4185 (2021). · DOI
  42. Scheres, S. H. RELION: implementation of a Bayesian approach to cryo-EM structure determination. J. Struct. Biol. 180, 519–530 (2012). · DOI · Paper page
  43. Zheng, S. Q. et al. MotionCor2: anisotropic correction of beam-induced motion for improved cryo-electron microscopy. Nat. Methods 14, 331–332 (2017). · DOI
  44. Rohou, A. & Grigorieff, N. CTFFIND4: Fast and accurate defocus estimation from electron micrographs. J. Struct. Biol. 192, 216–221 (2015). · DOI
  45. Wagner, T. et al. SPHIRE-crYOLO is a fast and accurate fully automated particle picker for cryo-EM. Commun. Biol. 2, 218 (2019). · DOI
  46. Zivanov, J., Nakane, T. & Scheres, S. H. W. A Bayesian approach to beam-induced motion correction in cryo-EM single-particle analysis. IUCrJ 6, 5–17 (2019). · DOI
  47. Goddard, T. D. et al. UCSF ChimeraX: Meeting modern challenges in visualization and analysis. Protein Sci. 27, 14–25 (2018). · DOI · Paper page
  48. Emsley, P. & Cowtan, K. Coot: Model-Building Tools for Molecular Graphics. Acta Crystallogr. Sect. D. - Biol. Crystallogr. 60, 2126–2132 (2004). · DOI · Paper page
  49. Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development of Coot. Acta Crystallogr D. Biol. Crystallogr 66, 486–501 (2010). · DOI · Paper page
  50. Winn, M. D. et al. Overview of the CCP4 suite and current developments. Acta Crystallogr D. Biol. Crystallogr 67, 235–242 (2011). · DOI · Paper page
  51. Pichkur, E. B. et al. Insights into the improved macrolide inhibitory activity from the high-resolution cryo-EM structure of dirithromycin bound to the E. coli 70S ribosome. RNA 26, 715–723 (2020). · DOI
  52. Yamashita, K., Palmer, C. M., Burnley, T. & Murshudov, G. N. Cryo-EM single-particle structure refinement and map calculation using Servalcat. Acta Crystallogr D. Struct. Biol. 77, 1282–1291 (2021). · DOI
  53. Long, F. et al. AceDRG: a stereochemical description generator for ligands. Acta Crystallogr D. Struct. Biol. 73, 112–122 (2017). · DOI
  54. Liebschner, D. et al. Macromolecular structure determination using X-rays, neutrons and electrons: recent developments in Phenix. Acta Crystallogr D. Struct. Biol. 75, 861–877 (2019). · DOI · Paper page
  55. Chen, V. B. et al. MolProbity: all-atom structure validation for macromolecular crystallography. Acta Crystallogr D. Biol. Crystallogr 66, 12–21 (2010). · DOI · Paper page

Metadata

Authors
Safdari, H. A., Morici, M., Sanchez-Castro, A., Dallapè, A., Paternoga, H., Giuliodori, A. M., Fabbretti, A., Milón, P., & Wilson, D. N.
Affiliations
  • Universität Hamburg (DE)
  • Peruvian University of Applied Sciences (PE)
  • Università di Camerino (IT)
Corresponding author
  • Milón, Pohl
  • Wilson, Daniel N.
ORCID
Venue
Nature Communications
Publisher
Springer Science and Business Media LLC
Published
2025-03-12
Volume
16
Issue
1
Pages
2470-2470
ISSN
2041-1723 (ISSN-L)
DOI
10.1038/s41467-025-57731-8
PMC
PMC11903750
OpenAlex
W4408360839
Cited by
11
References
3
Citations by year
2026: 7 · 2025: 4
FWCI
3.28
Type
journal article
Language
en
Open access
Licence
Locations
  • Nature Communications (publishedVersion, cc-by)
  • bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion, cc-by-nc-nd)
  • Europe PMC (PubMed Central) (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • LA Referencia (Red Federada de Repositorios Institucionales de Publicaciones Científicas) (submittedVersion, cc-by)
  • Unicam Scientific Publications (University of Camerino) (submittedVersion, cc-by)
  • RePEc: Research Papers in Economics (submittedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
  • Zenodo (CERN European Organization for Nuclear Research) (submittedVersion, cc-by)
Funders
Deutsche Forschungsgemeinschaft (WI3285/12-1)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA and protein synthesis mechanisms
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA modifications and cancer
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Bacterial Genetics and Biotechnology
Keywords
translation initiation, 30S ribosomal subunit, kasugamycin, edeine, translation inhibitors, preinitiation complex, start codon recognition, cryo-EM structure, antibiotic mechanism of action
MeSH terms
Aminoglycosides, Anti-Bacterial Agents, Escherichia coli, Peptide Chain Initiation, Translational, Protein Synthesis Inhibitors, Protein Biosynthesis, Codon, Initiator, Cryoelectron Microscopy, Ribosome Subunits, Small, Bacterial

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