GSE185757
The context of the ribosome binding site in mRNAs defines specificity of action of kasugamycin, an inhibitor of translation initiation
Kasugamycin (KSG) is an aminoglycoside antibiotic widely used in agriculture and exhibiting considerable medical potential. Previous studies suggested that KSG interferes with translation by blocking binding of canonical mRNA and initiator tRNA to the small ribosomal subunit thereby preventing initiation of protein synthesis. Here, by using genome-wide approaches, we show that KSG can interfere with translation even after the formation of the 70S initiation complex on mRNA, as the extent of KSG-mediated translation inhibition correlates with increased occupancy of start codons by 70S ribosomes. We also show that KSG inhibits protein synthesis in a gene- and context-specific manner as even saturating concentrations of KSG do not completely abolish translation of all Escherichia coli genes. Differential action of KSG significantly depends on the nature of the mRNA residue immediately preceding the start codon, with guanine in this position being the most conducive to inhibition by the drug.  In addition, the activity of KSG is attenuated by translational coupling as genes whose start codons overlap with the coding regions or the stop codons of the upstream cistrons tend to be less susceptible to drug-mediated inhibition. Altogether, our findings reveal KSG as the first example of a small ribosomal subunit-targeting antibiotic with a well-pronounced context specificity of action. Overall design: Ribosome profiling (Ribo-seq) and mRNA-seq data collected from E. coli ?gcvB cells grown in MOPS EZ rich defined media at mid-log phase, and the cells treated with 1 mg/ml an 10 mg/ml kasugamycin (KSG) for 2.5 min at 37°C.