Retapamulin-Assisted Ribosome Profiling Reveals the Alternative Bacterial Proteome

The use of alternative translation initiation sites enables production of more than one protein from a single gene, thereby expanding the cellular proteome. Although several such examples have been serendipitously found in bacteria, genome-wide mapping of alternative translation start sites has been unattainable. We found that the antibiotic retapamulin specifically arrests initiating ribosomes at start codons of the genes. Retapamulin-enhanced Ribo-seq analysis (Ribo-RET) not only allowed mapping of conventional initiation sites at the beginning of the genes, but strikingly, it also revealed putative internal start sites in a number of Escherichia coli genes. Experiments demonstrated that the internal start codons can be recognized by the ribosomes and direct translation initiation in vitro and in vivo. Proteins, whose synthesis is initiated at internal in-frame and out-of-frame start sites, can be functionally important and contribute to the “alternative” bacterial proteome. The internal start sites may also play regulatory roles in gene expression. [Display omitted] •Retapamulin arrests bacterial ribosomes specifically at translation start sites•Retapamulin-assisted Ribo-seq reveals known and cryptic translation start sites•Translation from start sites located within the ORFs may generate functional proteins•Start-stop sites found within some genes may help to regulate gene expression Meydan et al. report that the antibiotic retapamulin arrests bacterial ribosomes specifically at start codons. Retapamulin-assisted ribosome profiling detects known start sites but also reveals multiple cryptic sites within or outside the coding regions. Translation from internal start sites may lead to generation of functional proteins or play a regulatory role.