Structural and functional characterization of the bacterial translocation inhibitor GE82832

► Elucidation of the molecular target and mechanism of action of the antibiotic dityromycin. ► Elucidation of the structure of the antibiotic GE82832. ► Demonstration that dityromycin and GE82832 are almost identical inhibitors of the unlocking step of translocation. The structure of GE82832, a translocation inhibitor produced by a soil microorganism, is shown to be highly related to that of dityromycin, a bicyclodecadepsipeptide antibiotic discovered long ago whose characterization had never been pursued beyond its structural elucidation. GE82832 and dityromycin were shown to interfere with both aminoacyl-tRNA and mRNA movement and with the Pi release occurring after ribosome- and EF-G-dependent GTP hydrolysis. These findings and the unusual ribosomal localization of GE82832/dityromycin near protein S13 suggest that the mechanism of inhibition entails an interference with the rotation of the 30S subunit “head” which accompanies the ribosome-unlocking step of translocation.