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A rapid, simple, and economical method for the isolation of ribosomes and translational machinery for structural and functional studies

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Listed:
  • Jessey Erath

    (Washington University School of Medicine)

  • Danielle Kemper

    (Washington University School of Medicine)

  • Elisha Mugo

    (Washington University School of Medicine)

  • Alex Jacoby

    (Washington University School of Medicine)

  • Elizabeth Valenzuela

    (Columbia University)

  • Courtney F. Jungers

    (Washington University School of Medicine)

  • Wandy L. Beatty

    (Washington University School of Medicine)

  • Yaser Hashem

    (Université de Bordeaux)

  • Marko Jovanovic

    (Columbia University)

  • Sergej Djuranovic

    (Washington University School of Medicine
    Brown University
    Institute for Interdisciplinary and Multidisciplinary Studies)

  • Slavica Pavlovic Djuranovic

    (Washington University School of Medicine)

Abstract

Ribosomes are RNA-protein complexes essential for protein synthesis and quality control. Traditional methods for ribosome isolation are labor-intensive, expensive, and require a substantial amount of biological material. In contrast, our method, RNA affinity purification using poly-lysine (RAPPL), provides a rapid, simple, and cost-effective alternative applicable to various species and types of starting material (cell lysates, whole cells, organs, or whole organisms). It is also compatible with traditional isolation techniques. Here, we describe the use of RAPPL for rapid isolation, functional screening, and structural analysis of ribosomes and associated factors. We also demonstrate the application of RAPPL in investigating ribosome-associated resistance mechanisms in uropathogenic Escherichia coli samples and generating a 2.7-Å cryoEM ribosome structure from Cryptococcus neoformans. By significantly reducing the amount of the starting biological material and the time required for isolation, RAPPL has the potential to facilitate the study of ribosomal function, interactions, and antibiotic resistance and provide a versatile platform for academic, clinical, and industrial research.

Suggested Citation

  • Jessey Erath & Danielle Kemper & Elisha Mugo & Alex Jacoby & Elizabeth Valenzuela & Courtney F. Jungers & Wandy L. Beatty & Yaser Hashem & Marko Jovanovic & Sergej Djuranovic & Slavica Pavlovic Djuran, 2025. "A rapid, simple, and economical method for the isolation of ribosomes and translational machinery for structural and functional studies," Nature Communications, Nature, vol. 16(1), pages 1-16, December.
    • Handle: RePEc:nat:natcom:v:16:y:2025:i:1:d:10.1038_s41467-025-62314-8
    DOI: 10.1038/s41467-025-62314-8
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    References listed on IDEAS

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    1. T. Martin Schmeing & V. Ramakrishnan, 2009. "What recent ribosome structures have revealed about the mechanism of translation," Nature, Nature, vol. 461(7268), pages 1234-1242, October.
    2. Cédric Orelle & Erik D. Carlson & Teresa Szal & Tanja Florin & Michael C. Jewett & Alexander S. Mankin, 2015. "Protein synthesis by ribosomes with tethered subunits," Nature, Nature, vol. 524(7563), pages 119-124, August.
    3. Yaser Hashem & Amedee des Georges & Jie Fu & Sarah N. Buss & Fabrice Jossinet & Amy Jobe & Qin Zhang & Hstau Y. Liao & Robert A. Grassucci & Chandrajit Bajaj & Eric Westhof & Susan Madison-Antenucci &, 2013. "High-resolution cryo-electron microscopy structure of the Trypanosoma brucei ribosome," Nature, Nature, vol. 494(7437), pages 385-389, February.
    4. Rui Xu & Wandy L. Beatty & Valentin Greigert & William H. Witola & L. David Sibley, 2024. "Multiple pathways for glucose phosphate transport and utilization support growth of Cryptosporidium parvum," Nature Communications, Nature, vol. 15(1), pages 1-15, December.
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