Conformational Response of 30s-Bound IF3 to A-Site Binders Streptomycin and Kanamycin

Aminoglycoside antibiotics are widely used to treat infectious diseases. Among them, streptomycin and kanamycin (and derivatives) are of importance to battle multidrug-resistant (MDR) Mycobacterium tuberculosis. Both drugs bind the small ribosomal subunit (30S) and inhibit protein synthesis. Genetic, structural, and biochemical studies indicate that local and long-range conformational rearrangements of the 30S subunit account for this inhibition. Here, we use intramolecular FRET between the C- and N-terminus domains of the flexible IF3 to monitor real-time perturbations of their binding sites on the 30S platform. Steady and pre-steady state binding experiments show that both aminoglycosides bring IF3 domains apart, promoting an elongatedstateofthefactor. BindingofInitiationFactorIF1triggersclosureofIF3boundtothe30S complex,whilebothaminoglycosidesreverttheIF1-dependentconformation. Ourresultsuncover dynamicperturbationsacrossthe30Ssubunit,fromtheA-sitetotheplatform,andsuggestthatboth aminoglycosidescouldinterferewithprokaryotictranslationinitiationbymodulatingtheinteraction betweenIF3domainswiththe30Splatform.