Molecular basis of RNase I-mediated rRNA degradation regulated by ribosomes

Protein biosynthesis is an energy-hungry intracellular process that requires the stringent regulation of ribosome abundance under environmental conditions. In response to stress, some active ribosomes are degraded while others, in bacteria, enter a hibernation state to protect against degradation. RNase I, a conserved T2 family ribonuclease in Escherichia coli , degrades ribosomal RNA to suppress biofilm formation, whereas it interacts with ribosomes. However, how and why RNase I binds to ribosomes remains elusive. Here, we show that hibernating ribosomes bind to RNase I and inhibit its activity, thereby promoting biofilm formation. We determined the cryo-electron microscopy structure of the hibernating ribosome complexed with RNase I. RNase I interacts with helix 41 of the 16S rRNA and the ribosomal protein uS14 in the head domain of the 30S subunit of the hibernating ribosome and positions its active centre away from helix 41, resulting in its catalytic inactivation. Hibernating ribosomes are protected from RNase I-mediated cleavage, and our in vivo and in vitro analyses revealed that RNase I targets dissociated large and small ribosomal subunits for rRNA degradation. These findings reveal a previously uncharacterized regulatory strategy that ribosomes modulate RNase I activity, ensuring both the preservation and timely degradation of ribosomes during environmental stress adaptation.