Project Summary Bacteria respond to zinc starvation by replacing ribosomal proteins that have the zinc- binding CXXC motif (C+) with their zinc-free (C-) paralogs. Consequence of this process is generally known to balance zinc homeostasis under depletion of the micronutrient. Recently, we observed that the C- ribosome in Mycobacterium smegmatis is the exclusive target of a protein Y homolog, called mycobacterial protein Y (MPY). The cryo- EM structure reveals that MPY binds to the decoding region of the 30S subunit, thereby inactivating the ribosome. Moreover, MPY binding is dependent on another mycobacterial protein, called MPY recruitment factor (MRF), which is induced upon zinc depletion and interacts with C- ribosomes. Binding of MPY confers structural stability to C- ribosomes, thereby increasing viability of growth-arrested M. smegmatis under zinc- limiting conditions. Furthermore expression of C- ribosomes was observed in Mycobacterium tuberculosis during infection, leading to a hypothesis the zinc-depleted C- ribosome in the tuberculosis pathogen is also a target of the MPY homologue in this species. The fundamental implication of our hypothesis is that zinc depletion induces metabolic dormancy in M. tuberculosis. We will test this hypothesis by determining the binding of MPY homologue of M. tuberculosis ribosomes produced under zinc starvation (Aim 1). To obtain a direct correlation between ribosome hibernation and metabolic dormancy, we will develop a method to visualize ribosome hibernation in live mycobacterial cells (Aim 2). Together, the proposal will develop a molecular framework for a long-term investigation on ribosome hibernation and dormancy in M. tuberculosis.