Abstract
Kasugamycin (KSG) is an aminoglycoside antibiotic widely used in agriculture and exhibits considerable medical potential. Previous studies suggested that KSG interferes with translation by blocking binding of canonical messenger RNA (mRNA) and initiator transfer tRNA (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. Even at saturating concentrations, KSG does not completely abolish translation, allowing for continuing expression of some Escherichia coli proteins. 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 an example of a small ribosomal subunit-targeting antibiotic with a well-pronounced context specificity of action.
Highlights
Kasugamycin (KSG) is an aminoglycoside antibiotic widely used in agriculture and exhibits considerable medical potential
We show that kasugamycin-induced ribosomal arrest at the start codons of the genes and the resulting inhibition of gene expression depend on the nature of the messenger RNA (mRNA) nucleotide immediately preceding the start codon and on the proximity of the stop codon of the upstream cistron
Since the ΔgcvB mutant is characterized by a faster uptake of the drug [38], we were able to reduce the time of antibiotic treatment at 1,000× minimal inhibitory concentration (MIC) to 2.5 min to minimize the effects of secondary responses induced by persistent translation inhibition while still achieving the plateau levels of residual translation (SI Appendix, Fig. S2A)
Summary
Kasugamycin (KSG) is an aminoglycoside antibiotic widely used in agriculture and exhibits considerable medical potential. Previous studies suggested that KSG interferes with translation by blocking binding of canonical messenger RNA (mRNA) and initiator transfer tRNA (tRNA) to the small ribosomal subunit, thereby preventing initiation of protein synthesis. 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. Our findings reveal KSG as an example of a small ribosomal subunit-targeting antibiotic with a wellpronounced context specificity of action. We reveal kasugamycin as a small ribosomal subunit-targeting antibiotic whose action depends on the sequence context of the untranslated messenger RNA (mRNA) segments. Our findings underlie the importance of mRNA context for the action of protein synthesis inhibitors and might help to guide the development of better antibiotics. Initiation of protein synthesis involves a stepwise assembly of the translation complex at the start codon of an open reading frame (ORF) (reviewed in Refs. 5 and 6)
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