Abstract
In bacteria, ribosomal hibernation shuts down translation as a response to stress, through reversible binding of stress-induced proteins to ribosomes. This process typically involves the formation of 100S ribosome dimers. Here, we present the structures of hibernating ribosomes from human pathogen Staphylococcus aureus containing a long variant of the hibernation-promoting factor (SaHPF) that we solved using cryo-electron microscopy. Our reconstructions reveal that the N-terminal domain (NTD) of SaHPF binds to the 30S subunit as observed for shorter variants of HPF in other species. The C-terminal domain (CTD) of SaHPF protrudes out of each ribosome in order to mediate dimerization. Using NMR, we characterized the interactions at the CTD-dimer interface. Secondary interactions are provided by helix 26 of the 16S ribosomal RNA We also show that ribosomes in the 100S particle adopt both rotated and unrotated conformations. Overall, our work illustrates a specific mode of ribosome dimerization by long HPF, a finding that may help improve the selectivity of antimicrobials.