Context specificity of translation inhibitors revealed by toe-seq

Translation is a critical step in gene expression and a target for multiple antibiotics. Inhibition of protein synthesis by many antibiotics depends on mRNA context, making them selective towards particular mRNAs. Toe-printing is a standard low-throughput method to determine ribosome position along mRNA. Hereby we report the development and application of the Toe-seq method, combining toe-printing with next-generation sequencing. Toe-seq proved to be efficient to monitor translation and its inhibition by antibiotics for the library of over 37 000 mRNAs. Context-specificity of antibiotic action could be determined for different antibiotics via Toe-seq. Previously known sequence dependencies of translation inhibition were reproduced by Toe-seq. Data obtained by Toe-seq demonstrated context-specificity of etamycin A (EtaA) action whose binding site is located in the nascent peptide tunnel. Comparison of EtaA specificity with that of erythromycin (Ery) and tetracenomycin X (TcmX) revealed important differences and commonalities. Structural basis for the context-specificity of translation inhibition by EtaA has been deciphered by cryo-electron microscopy at 2.2 Å resolution. Toe-seq method allows one to monitor ribosome progression and stalling in vitro at a codon resolution, making it possible to assess antibiotics context specificity in a multiple parallel assay.