Roles of specific aminoglycoside–ribosome interactions in the inhibition of translation

Aminoglycosides containing a 2-deoxystreptamine core (AGs) represent a large family of antibiotics that target the ribosome. These compounds promote miscoding, inhibit translocation, and inhibit ribosome recycling. AG binding to helix h44 of the small subunit induces rearrangement of A-site nucleotides A1492 and A1493, which promotes a key open-to-closed conformational change of the subunit and thereby increases miscoding. Mechanisms by which AGs inhibit translocation and recycling remain less clear. Structural studies have revealed a secondary AG binding site in H69 of the large subunit, and it has been proposed that interaction at this site is crucial for inhibition of translocation and recycling. Here, we analyze ribosomes with mutations targeting either or both AG binding sites. Assaying translocation, we find that ablation of the h44 site increases the IC(50) values for AGs dramatically, while removal of the H69 site increases these values modestly. This suggests that the AG–h44 interaction is primarily responsible for inhibition, with H69 playing a minor role. Assaying recycling, we find that mutation of h44 has no effect on AG inhibition, consistent with a primary role for AG–H69 interaction. Collectively, these findings help clarify the roles of the two AG binding sites in mechanisms of inhibition by these compounds.