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Aminoglycosides are widely used antibiotics that cause messenger RNA decoding errors, block mRNA and transfer RNA translocation, and inhibit ribosome recycling. Ribosome recycling follows the termination of protein synthesis and is aided by ribosome recycling factor (RRF) in bacteria. The molecular mechanism by which aminoglycosides inhibit ribosome recycling is unknown. Here we show in X-ray crystal structures of the Escherichia coli 70S ribosome that RRF binding causes RNA helix H69 of the large ribosomal subunit, which is crucial for subunit association, to swing away from the subunit interface. Aminoglycosides bind to H69 and completely restore the contacts between ribosomal subunits that are disrupted by RRF. These results provide a structural explanation for aminoglycoside inhibition of ribosome recycling.

The prokaryotic ribosome is an important target of antibiotic action. We determined the X-ray structure of the aminoglycoside kasugamycin (Ksg) in complex with the Escherichia coli 70S ribosome at 3.5-A resolution. The structure reveals that the drug binds within the messenger RNA channel of the 30S subunit between the universally conserved G926 and A794 nucleotides in 16S ribosomal RNA, which are sites of Ksg resistance. To our surprise, Ksg resistance mutations do not inhibit binding of the drug to the ribosome. The present structural and biochemical results indicate that inhibition by Ksg and Ksg resistance are closely linked to the structure of the mRNA at the junction of the peptidyl-tRNA and exit-tRNA sites (P and E sites).

During environmental stress, organisms limit protein synthesis by storing inactive ribosomes that are rapidly reactivated when conditions improve. Here we present structural and biochemical data showing that protein Y, an Escherichia coli stress protein, fills the tRNA- and mRNA-binding channel of the small ribosomal subunit to stabilize intact ribosomes. Protein Y inhibits translation initiation during cold shock but not at normal temperatures. Furthermore, protein Y competes with conserved translation initiation factors that, in bacteria, are required for ribosomal subunit dissociation. The mechanism used by protein Y to reduce translation initiation during stress and quickly release ribosomes for renewed translation initiation may therefore occur widely in nature.