A ketolide resistance mutation in domain II of 23S rRNA reveals the proximity of hairpin 35 to the peptidyl transferase centre

Ketolides represent a new generation of macrolide antibiotics. In order to identify the ketolide-binding site on the ribosome, a library of Escherichi a coil clones, transformed with a plasmid carrying randomly mutagenized rRN A operon, was screened for mutants exhibiting resistance to the ketolide HM R3647, Sequencing of the plasmid isolated from one of the resistant clones and fragment exchange demonstrated that a single U754A mutation in hairpin 35 of domain II of the E. coli 23S rRNA was sufficient to confer resistance to low concentrations of the ketolide, The same mutation also conferred er ythromycin resistance, Both the ketolide and erythromycin protected A2058 a nd A2059 in domain V of 23S rRNA from modification with dimethyl sulphate, whereas, in domain II, the ketolide protected, while erythromycin enhanced, modification of A752 in the loop of the hairpin 35. Thus, mutational and f ootprinting results strongly suggest that the hairpin 35 constitutes part o f the macrolide binding site on the ribosome, Strong interaction of ketolid es with the hairpin 35 in 23S rRNA may account for the high activity of ket olides against erythromycin-resistant strains containing rRNA methylated at A2058, The existence of macrolide resistance mutations in the central loop of domain V and in hairpin 35 in domain II together with antibiotic footpr inting data suggest that these rRNA segments may be in close proximity in t he ribosome and that hairpin 35 may be a constituent part of the ribosomal peptidyl transferase centre.