Chapter 5
Purification of Hibernating and Active CÀ Ribosomes
from Zinc-Starved Mycobacteria
Yunlong Li, Pooja Keshavan, Jamie H. Corro, Ravi K. Koripella,
Rajendra K. Agrawal, and Anil K. Ojha
Abstract
Zinc starvation in Mycobacterium smegmatis and Mycobacterium tuberculosis induces ribosome remodeling
and hibernation. Remodeling involves replacement of C+ ribosomal (r-) proteins containing the zincbinding CXXC motif with their CÀ paralogues without the motif. Hibernation is characterized by binding
of mycobacterial-specific protein Y (Mpy) to 70S CÀ 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 CÀ 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.
Key words Mycobacterial persistence, Dug resistance, Dormancy
1 Introduction
In a growing Escherichia coli cell, about 40% of the total energy is
dedicated towards the protein synthesis [1], and its downregulation
during starvation would conceivably generate a sizable pool of
non-translating 70S ribosomes. The fate of these ribosomes
remains an emerging topic of investigation in the larger context
of deciphering the mechanisms of starvation adaptation and associated antibiotic tolerance in bacteria. Analyses of ribosomes from
stationary phase cells of both Gram-positive and Gram-negative
bacterial species have identified specialized proteins that preserve
ribosomes in an inactive 100S disomes or 70S monosomes, collectively termed hibernating ribosomes [2–7]. Proteins involved in formation of hibernating 100S disomes are commonly referred to as
Tanya Parish and Anuradha Kumar (eds.), Mycobacteria Protocols, Methods in Molecular Biology, vol. 2314,
https://doi.org/10.1007/978-1-0716-1460-0_5, © Springer Science+Business Media, LLC, part of Springer Nature 2021
151
Purification of Hibernating and Active CÀ Ribosomes
from Zinc-Starved Mycobacteria
Yunlong Li, Pooja Keshavan, Jamie H. Corro, Ravi K. Koripella,
Rajendra K. Agrawal, and Anil K. Ojha
Abstract
Zinc starvation in Mycobacterium smegmatis and Mycobacterium tuberculosis induces ribosome remodeling
and hibernation. Remodeling involves replacement of C+ ribosomal (r-) proteins containing the zincbinding CXXC motif with their CÀ paralogues without the motif. Hibernation is characterized by binding
of mycobacterial-specific protein Y (Mpy) to 70S CÀ 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 CÀ 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.
Key words Mycobacterial persistence, Dug resistance, Dormancy
1 Introduction
In a growing Escherichia coli cell, about 40% of the total energy is
dedicated towards the protein synthesis [1], and its downregulation
during starvation would conceivably generate a sizable pool of
non-translating 70S ribosomes. The fate of these ribosomes
remains an emerging topic of investigation in the larger context
of deciphering the mechanisms of starvation adaptation and associated antibiotic tolerance in bacteria. Analyses of ribosomes from
stationary phase cells of both Gram-positive and Gram-negative
bacterial species have identified specialized proteins that preserve
ribosomes in an inactive 100S disomes or 70S monosomes, collectively termed hibernating ribosomes [2–7]. Proteins involved in formation of hibernating 100S disomes are commonly referred to as
Tanya Parish and Anuradha Kumar (eds.), Mycobacteria Protocols, Methods in Molecular Biology, vol. 2314,
https://doi.org/10.1007/978-1-0716-1460-0_5, © Springer Science+Business Media, LLC, part of Springer Nature 2021
151