Retapamulin-assisted ribosome profiling reveals the alternative bacterial proteome

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Meydan, Sezen
Marks, James
Klepacki, Dorota
Sharma, Virag
Baranov, Pavel V.
Firth, Andrew E.
Margus, Tonu
Kefi, Amira
Vázquez-Laslop, Nora
Mankin, Alexander S.
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Elsevier Inc.
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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.
Ribosome profiling , Translation initiation , Retapamulin , Alternative initiation , Internal genes
Meydan, S., Marks, J., Klepacki, D., Sharma, V., Baranov, P. V., Firth, A. E., Margus, T., Kefi, A., Vázquez-Laslop, N. and Mankin, A. S. (2019) 'Retapamulin-assisted ribosome profiling reveals the alternative bacterial proteome', Molecular Cell, 74(3), pp. 481-493. doi: 10.1016/j.molcel.2019.02.017