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Purification of Hibernating and Active C− Ribosomes from Zinc-Starved Mycobacteria

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Mycobacteria Protocols

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2314))

Abstract

Zinc starvation in Mycobacterium smegmatis and Mycobacterium tuberculosis induces ribosome remodeling and hibernation. Remodeling involves replacement of C+ ribosomal (r-) proteins containing the zinc-binding CXXC motif with their C− paralogues without the motif. Hibernation is characterized by binding of mycobacterial-specific protein Y (Mpy) to 70S C− ribosomes, stabilizing the ribosome in an inactive state that is also resistant to kanamycin and streptomycin. We observed that ribosome remodeling and hibernation occur at two different concentrations of cellular zinc. Here, we describe the methods to purify hibernating and active forms of C− ribosomes from zinc-starved mycobacteria, along with purification of C+ ribosomes from zinc-rich mycobacterial cells. In vitro analysis of these distinct types of ribosomes will facilitate screening of small molecule inhibitors of ribosome hibernation for improved therapeutics against mycobacterial infections.

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Acknowledgments

Support from Tissue Culture and Media core facility of the Wadsworth Center in making the buffer stocks is acknowledged. Authors also acknowledge funding support from the National Institute of Health (AI132422 and AI144474 to A.K.O. and GM061576 to R.K.A.) and New York State Department of Health in carrying out this work.

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Authors and Affiliations

  1. Division of Genetics, Wadsworth Center, New York State Department of Health, Albany, NY, USA

    Yunlong Li, Jamie H. Corro & Anil K. Ojha

  2. Division of Translational Medicine, Wadsworth Center, New York State Department of Health, Albany, NY, USA

    Pooja Keshavan, Ravi K. Koripella & Rajendra K. Agrawal

  3. Department of Biomedical Sciences, University at Albany, Albany, NY, USA

    Jamie H. Corro, Rajendra K. Agrawal & Anil K. Ojha

Authors
  1. Yunlong Li
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  2. Pooja Keshavan
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  3. Jamie H. Corro
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  4. Ravi K. Koripella
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  5. Rajendra K. Agrawal
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  6. Anil K. Ojha
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Corresponding author

Correspondence to Anil K. Ojha .

Editor information

Editors and Affiliations

  1. Center for Global Infectious Disease Research, Seattle Children’s Research Institute, Seattle, WA, USA

    Tanya Parish

  2. Center for Global Infectious Disease Research, Seattle Children’s Research Institute, Seattle, WA, USA

    Anuradha Kumar

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Li, Y., Keshavan, P., Corro, J.H., Koripella, R.K., Agrawal, R.K., Ojha, A.K. (2021). Purification of Hibernating and Active C− Ribosomes from Zinc-Starved Mycobacteria. In: Parish, T., Kumar, A. (eds) Mycobacteria Protocols. Methods in Molecular Biology, vol 2314. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1460-0_5

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  • DOI: https://doi.org/10.1007/978-1-0716-1460-0_5

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-1459-4

  • Online ISBN: 978-1-0716-1460-0

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