Summary Changes in abundance of zinc, an essential nutrient but toxic at high concentrations, necessitates adaptive mechanisms in mycobacteria. Under zinc-rich conditions, mycobacteria synthesize ribosomes with a set of ribosomal (r-) proteins containing the zinc-binding CXXC (C+) motif, while zinc starvation induces paralogs of these proteins called C- (for the lack of CXXC motif) as replacements in the 70S ribosome. Evidence of C+ to C- ribosome remodeling is reported in Mtb from chronic lung infections in mice and humans. Clinical relevance of ribosome remodeling is supported by greater antibiotic resistance in the C- than C+ ribosomes, highlighting the challenges in using ribosome targeting drugs in TB therapy. Previously, we solved the structures of C+ and C- ribosomes to decipher the mechanism of their differential antibiotic resistance. We discovered the C- ribosome as the preferred target of mycobacterial protein Y (Mpy). Mpy is a member of highly conserved family of bacterial protein Y, which binds to the decoding region of the ribosome, thereby hibernating non- translating ribosomes as 70S monosomes in growth arrested cells. The structure identified two distinct drug resistance mechanisms associated with the C- ribosome. Aminoglycoside resistance is largely associated with the preferential binding of Mpy to the decoding center of the C- ribosome, which directly impacts the streptomycin and kanamycin binding pockets within the C- ribosome. Unlike aminoglycosides, tolerance to spectinamides is a direct consequence of the structural changes in the 30S ribosomal subunit due to C+ to C- substitution of the r-protein, S14. During our investigation of ribosome hibernation as a specific response to zinc starvation, we discovered a zinc-sensing Mpy- recruitment factor (Mrf), that ensures Mpy recruitment to C- ribosome. The mechanism by which Mpy is recruited to C- ribosomes, resulting in ribosome hibernation, remains unknown. In this proposal, we will specifically focus on: a) deciphering the mechanism of Mpy recruitment to the ribosome, and b) identifying the mechanistic basis for Mpy-dependent and -independent drug resistance in mycobacteria expressing C- ribosomes. The findings will expand our understanding of antibiotic tolerance associated with ribosome remodeling and hibernation in zinc-starved mycobacteria. Given that lung environment of Mtb in humans is likely zinc-limiting, our findings will inform and improve the therapeutic strategies for TB, possibly leading to a shorter treatment regimen.