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. 2021:2314:151-166.
doi: 10.1007/978-1-0716-1460-0_5.

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

Yunlong Li  1 , Pooja Keshavan  2 , Jamie H Corro  1   3 , Ravi K Koripella  2 , Rajendra K Agrawal  2   3 , Anil K Ojha  4   5
Affiliations

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

Yunlong Li et al. Methods Mol Biol. 2021.

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.

Keywords: Dormancy; Dug resistance; Mycobacterial persistence.

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Figures

Fig. 1
Fig. 1
Growth of M. smegmatis cells in Sauton’s medium base with 1 μM TPEN. Arrows indicate the timepoints at which saturating levels of remodeled and hibernating ribosomes were observed
Fig. 2
Fig. 2
A representative profile of ribosome fractionation on a 38.5 mL of 10–40% sucrose density gradient (SDG). Ribosomes equivalent to 100 U (OD260) were loaded on the SDG
Fig. 3
Fig. 3
Immunoblot analysis of 2.4 picomoles of C+ (96 h of growth in high-Zn Sauton’s medium; indicated by the absence of S14c− protein), active C− (28 h of growth in Sauton’s medium with TEPN; indicated by the presence of S14c− protein) and hibernating (Mpy-bound) ribosomes (96 h of growth in Sauton’s medium with TEPN). S13 was probed to normalize the loading amounts
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