Regulation and function of bacterial hibernating 100S ribosome

PROJECT SUMMARY Ribosome hibernation is a conserved mechanism used by both bacteria and eukaryotes to prevent translation and to extend organismal lifespan. Recent studies from various bacterial species, including pathogenic Staphylococcus aureus, have provided compelling evidence for a critical role of hibernating 100S ribosomes in protecting the ribosomal pool from damage, in addition to blocking translational initiation. We found that S. aureus ribosomes lacking hibernation-promoting factor (HPF) are rapidly degraded by the 3’-5’ exonuclease RNase R and other hitherto unknown ribonucleases. In our unpublished work, we isolated an additional ribonuclease mutant that rescues the loss of ribosomes in S. aureus. Surprisingly, we found that ribosomes are not the only target of S. aureus HPF; instead, HPF could interact with a cytoplasmic protein of unknown biological activity, thereby reducing the abundance of hibernating 100S ribosomes. We further demonstrated that HPF is restricted to a specific subcellular localization during rapid growth, providing a rare glimpse of possible HPF segregation from actively translating ribosomes. In this proposal, we will undertake a highly multidisciplinary approach consisting of structural biology, omics, bacterial genetics, biochemistry and high-resolution microscopy to achieve the following goals: (1) Determine the molecular mechanisms by which HPF protects ribosomes from ribonucleolytic cleavage. (2) Determine the previously undiscovered extraribosomal role of HPF. (3) Determine how the spatiotemporal localization of HPF avoids translation conflicts. HPF and RNase R are evolutionarily conserved virulence factors among nosocomial gram-positive and gram-negative bacteria, completion of these aims will provide significant mechanistic insight into innovative counterstrategies to combat recalcitrant infections by perturbing the biogenesis and turnover of hibernating ribosomes.