hibernation promoting factor (Hpf), while those forming hibernating 70S monosomes have been identified as RaiA (aka YfiA in
Escherichia coli) or pY-like proteins. In both cases, hibernating
proteins occupy the mRNA–tRNA interaction sites on the ribosome, encompassing the decoding center of the 30S subunit at the
subunit interface, thereby inhibiting the binding of canonical
ligands of protein synthesis including mRNA, tRNA, IF1, IF3,
and EF-G [8–19]. However, mechanisms regulating the recruitment of Hpf and pY-like proteins remain unclear in most bacterial
species.
We recently reported that hibernation of 70S ribosomes in
Mycobacterium smegmatis is induced under the specific condition
of zinc starvation by recruitment of mycobacterial protein-Y (Mpy)
near the decoding center [12]. The cryo-EM structure revealed
that Mpy recruitment also obstructs the conformational changes
needed for the binding of kanamycin and streptomycin on the
ribosome [12]. Growth-arrested M. smegmatis cells harboring
saturating levels of Mpy-bound ribosomes remain viable for
extended periods, and in a kanamycin and streptomycin tolerant
state [12].
Interestingly, zinc depletion also induces ribosome remodeling,
in which C+ ribosomal proteins containing the zinc-binding CXXC
motif are replaced by their CÀ paralogues without the motif
[12, 20, 26]. Furthermore, remodeling of C+ to CÀ ribosomes
occurs at higher cellular zinc levels than that inducing Mpy recruitment to CÀ ribosomes [21]. Importantly, Mpy-dependent ribosome hibernation was also observed in zinc-starved Mycobacterium
tuberculosis [21]. Further significance of these findings is highlighted by the evidence of CÀ ribosome expression [12], and
Mpy-dependent streptomycin resistance [21], in M. tuberculosis
during infection. Taken together, these data show a conserved
and biphasic pattern of zinc-responsive changes in mycobacterial
ribosomes—first remodeling of C+ to CÀ form, followed by
Mpy-dependent hibernation of CÀ ribosomes. Moreover, formation of hibernating CÀ ribosomes could possibly support persistence of mycobacteria in a growth-arrested and drug-tolerant
state [27]. Further structure–function studies with purified forms
of these types of ribosomes will provide new insights into similarities and differences between their properties and will allow the
development of suitable assays towards the discovery of small
molecules that inhibit ribosome hibernation. This chapter offers a
detailed protocol of purifying these types of ribosomes from both
Mycobacterium smegmatis and Mycobacterium tuberculosis. The purified ribosomes can be used for multiple downstream applications,
including cryo-EM structure analysis and in vitro translation assays.
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Escherichia coli) or pY-like proteins. In both cases, hibernating
proteins occupy the mRNA–tRNA interaction sites on the ribosome, encompassing the decoding center of the 30S subunit at the
subunit interface, thereby inhibiting the binding of canonical
ligands of protein synthesis including mRNA, tRNA, IF1, IF3,
and EF-G [8–19]. However, mechanisms regulating the recruitment of Hpf and pY-like proteins remain unclear in most bacterial
species.
We recently reported that hibernation of 70S ribosomes in
Mycobacterium smegmatis is induced under the specific condition
of zinc starvation by recruitment of mycobacterial protein-Y (Mpy)
near the decoding center [12]. The cryo-EM structure revealed
that Mpy recruitment also obstructs the conformational changes
needed for the binding of kanamycin and streptomycin on the
ribosome [12]. Growth-arrested M. smegmatis cells harboring
saturating levels of Mpy-bound ribosomes remain viable for
extended periods, and in a kanamycin and streptomycin tolerant
state [12].
Interestingly, zinc depletion also induces ribosome remodeling,
in which C+ ribosomal proteins containing the zinc-binding CXXC
motif are replaced by their CÀ paralogues without the motif
[12, 20, 26]. Furthermore, remodeling of C+ to CÀ ribosomes
occurs at higher cellular zinc levels than that inducing Mpy recruitment to CÀ ribosomes [21]. Importantly, Mpy-dependent ribosome hibernation was also observed in zinc-starved Mycobacterium
tuberculosis [21]. Further significance of these findings is highlighted by the evidence of CÀ ribosome expression [12], and
Mpy-dependent streptomycin resistance [21], in M. tuberculosis
during infection. Taken together, these data show a conserved
and biphasic pattern of zinc-responsive changes in mycobacterial
ribosomes—first remodeling of C+ to CÀ form, followed by
Mpy-dependent hibernation of CÀ ribosomes. Moreover, formation of hibernating CÀ ribosomes could possibly support persistence of mycobacteria in a growth-arrested and drug-tolerant
state [27]. Further structure–function studies with purified forms
of these types of ribosomes will provide new insights into similarities and differences between their properties and will allow the
development of suitable assays towards the discovery of small
molecules that inhibit ribosome hibernation. This chapter offers a
detailed protocol of purifying these types of ribosomes from both
Mycobacterium smegmatis and Mycobacterium tuberculosis. The purified ribosomes can be used for multiple downstream applications,
including cryo-EM structure analysis and in vitro translation assays.
152
Yunlong Li et al.