GSE193270
Context-specific translation arrest by evernimicin, an inhibitor of tRNA accommodation
Antibiotics of the orthosomycin class bind at a distinct site on the large subunit of the bacterial ribosome not used by any other known protein synthesis inhibitor. Structural and biochemical in vitro studies suggested that orthosomycins should block accommodation of aminoacyl-tRNAs in the ribosomal A-site arresting the ribosome at the start codons of the genes. However, the mode of action of orthosomycins in the living cell remains unknown. Here, to get a general and unbiased view of the mode of action of orthosomycin antibiotics, we carried out genome-wide ribosome profiling analysis in Escherichia coli cells exposed to evernimicin, one of the most active antibiotics of this class. Our in vivo data, supported by the analysis of evernimicin action upon in vitro translation of a variety of mRNAs, argue that orthosomycins preferentially inhibit translation elongation and act in a context specific manner. We show that evernimicin predominantly arrests translation when the ribosome needs to accommodate Pro-tRNA or Leu-tRNA in the A site while polymerizing specific amino acid sequences. We further show that the discovered context specificity of orthosomycins is exploited for the programmed translation arrest that apparently regulates resistance to these antibiotics. Overall design: Ribosome profiling of E. coli imp4213 cell cultures treated with evernimicin were compared to untreated cultures to study the mechanism of translation inhibition