Highlights d Retapamulin arrests bacterial ribosomes specifically at translation start sites d Retapamulin-assisted Ribo-seq reveals known and cryptic translation start sites d Translation from start sites located within the ORFs may generate functional proteins d Start-stop sites found within some genes may help to regulate gene expression

Additional informationPeer review information Nature thanks the anonymous reviewers for their contribution to the peer review of this work.

Highlights d Rocaglates produce distinct inhibitory effects on translation initiation d Rocaglates interfere with eIF4F release from the cap structure d Rocaglates exert a bystander effect on translation initiation by sequestering eIF4F

Phosphorylation of the S6 protein of the 40S subunit of the eukaryote ribosome downstream of anabolic signals has long been assumed to promote protein synthesis. Both target of rapamycin complexes regulate this modification in yeast, but the use of ribosome profiling shows no role for Rps6 phosphorylation in mRNA translation.