Starvation sensing by mycobacterial RelA/SpoT homologue through constitutive surveillance of translation.

The stringent response, which leads to persistence of nutrient-starved mycobacteria, is induced by activation of the RelA/SpoT homolog (Rsh) upon entry of a deacylated-tRNA in a translating ribosome. However, the mechanism by which Rsh identifies such ribosomes in vivo remains unclear. Here, we show that conditions inducing ribosome hibernation result in loss of intracellular Rsh in a Clp protease-dependent manner. This loss is also observed in nonstarved cells using mutations in Rsh that block its interaction with the ribosome, indicating that Rsh association with the ribosome is important for Rsh stability. The cryo-EM structure of the Rsh-bound 70S ribosome in a translation initiation complex reveals unknown interactions between the ACT domain of Rsh and components of the ribosomal L7/L12 stalk base, suggesting that the aminoacylation status of A-site tRNA is surveilled during the first cycle of elongation. Altogether, we propose a surveillance model of Rsh activation that originates from its constitutive interaction with the ribosomes entering the translation cycle.

Progression from remodeling to hibernation of ribosomes in zinc-starved mycobacteria.

Zinc starvation in mycobacteria leads to remodeling of ribosomes, in which multiple ribosomal (r-) proteins containing the zinc-binding CXXC motif are replaced by their motif-free paralogues, collectively called C- r-proteins. We previously reported that the 70S C- ribosome is exclusively targeted for hibernation by mycobacterial-specific protein Y (Mpy), which binds to the decoding center and stabilizes the ribosome in an inactive and drug-resistant state. In this study, we delineate the conditions for ribosome remodeling and hibernation and provide further insight into how zinc depletion induces Mpy recruitment to C- ribosomes. Specifically, we show that ribosome hibernation in a batch culture is induced at an approximately two-fold lower cellular zinc concentration than remodeling. We further identify a growth phase in which the C- ribosome remains active, while its hibernation is inhibited by the caseinolytic protease (Clp) system in a zinc-dependent manner. The Clp protease system destabilizes a zinc-bound form of Mpy recruitment factor (Mrf), which is stabilized upon further depletion of zinc, presumably in a zinc-free form. Stabilized Mrf binds to the 30S subunit and recruits Mpy to the ribosome. Replenishment of zinc to cells harboring hibernating ribosomes restores Mrf instability and dissociates Mpy from the ribosome. Finally, we demonstrate zinc-responsive binding of Mpy to ribosomes in Mycobacterium tuberculosis (Mtb) and show Mpy-dependent antibiotic tolerance of Mtb in mouse lungs. Together, we propose that ribosome hibernation is a specific and conserved response to zinc depletion in both environmental and pathogenic mycobacteria.

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

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.