High-throughput inverse toeprinting reveals the complete sequence dependence of ribosome-targeting antibiotics

It has recently become clear that various antibiotics block the translation of bacterial proteins in a sequence-specific manner. In order to understand how this specificity contributes to antibiotic potency and select better antimicrobial leads, new high-throughput tools are needed. Here, we present inverse toeprinting, a new method to map the position of ribosomes arrested on messenger RNAs during in vitro translation. Unlike ribosome profiling, our method protects the entire coding region upstream of a stalled ribosome, making it possible to work with transcript libraries that randomly sample the sequence space. We used inverse toeprinting to characterize the pausing landscape of free and drug-bound bacterial ribosomes engaged in translation. We obtained a comprehensive list of arrest motifs that could be validated in vivo , along with a quantitative measure of their pause strength. Thus, our method provides a highly parallel and scalable means to characterize the sequence specificity of translation inhibitors.