SHAPE footprinting as complementary approach to structure‐based design of ribosomal antibiotics: Phenicol antibiotics prevent A2451 2′OH acylation

Abstract

Early biochemical experiments suggested that phenyl propanoid antibiotics, particularly chloramphenicol (CAM), had more than one binding site on ribosomes. Indeed recent X‐ray structures have shown two distinct, non‐overlapping, binding sites for CAM bound to 50S ribosomal subunits. The structure of CAM bound to the 50S ribosomal subunit of Haloarcula marismortui shows an overlap of the CAM binding site with that of macrolide antibiotics (e.g. erythromycin, etc). Whereas the structure of CAM bound to Deinococcus radiodurans 50S ribosomal subunits shows an overlaps between the binding site of CAM with that of puromycin or the CCA‐end of A‐site tRNA. We have used SHAPE footprinting to map the location, in solution, of antibiotics, including several novel phenicol analogs, onto 70S ribosomes from Escherichia coli and Staphylococcus aureus. Our data clearly indicate that phenyl propanoid antibiotics are able to simultaneously occupy both binding sites in E. coli as well as in S. aureus ribosomes. Nevertheless, "macrolide‐like" footprints were less reproducible than "puromycin‐like" (A‐site) footprints. We found that phenicol binding to the "puromycin" binding site blocks 2′‐OH hydroxyl modification of nucleotide A2451. Interestingly, this hydroxyl group has been shown to be critical in peptide bond formation, thus our results are shedding more light on the mechanism of phenyl propanoid antibiotic action.