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Identifying small proteins by ribosome profiling with stalled initiation complexes

Jeremy Weaver, Fuad Mohammad, Allen R. Buskirk, View ORCID ProfileGisela Storz
doi: https://doi.org/10.1101/511675
Jeremy Weaver
a, Bethesda, MD 20892-5430, USA
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Fuad Mohammad
bDepartment of Molecular Biology and Genetics, Johns Hopkins School of Medicine, Baltimore, MD 21205, USA
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Allen R. Buskirk
bDepartment of Molecular Biology and Genetics, Johns Hopkins School of Medicine, Baltimore, MD 21205, USA
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Gisela Storz
a, Bethesda, MD 20892-5430, USA
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ABSTRACT

Small proteins consisting of 50 or fewer amino acids have been identified as regulators of larger proteins in bacteria and eukaryotes. Despite the importance of these molecules, the true prevalence of small proteins remains unknown because conventional annotation pipelines usually exclude small open reading frames (smORFs). We previously identified several dozen small proteins in the model organism Escherichia coli using theoretical bioinformatic approaches based on sequence conservation and matches to canonical ribosome binding sites. Here, we present an empirical approach for discovering new proteins, taking advantage of recent advances in ribosome profiling in which antibiotics are used to trap newly-initiated 70S ribosomes at start codons. This approach led to the identification of many novel initiation sites in intergenic regions in E. coli. We tagged 41 smORFs on the chromosome and detected protein synthesis for all but three. The corresponding genes are not only intergenic, but are also found antisense to other genes, in operons, and overlapping other open reading frames (ORFs), some impacting the translation of larger downstream genes. These results demonstrate the utility of this method for identifying new genes, regardless of their genomic context.

IMPORTANCE Proteins comprised of 50 or fewer amino acids have been shown to interact with and modulate the function of larger proteins in a range of organisms. Despite the possible importance of small proteins, the true prevalence and capabilities of these regulators remain unknown as the small size of the proteins places serious limitations on their identification, purification and characterization. Here, we present a ribosome profiling approach with stalled initiation complexes that led to the identification of 38 new small proteins.

Copyright 
The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. This article is a US Government work. It is not subject to copyright under 17 USC 105 and is also made available for use under a CC0 license.
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Posted January 04, 2019.
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Identifying small proteins by ribosome profiling with stalled initiation complexes
Jeremy Weaver, Fuad Mohammad, Allen R. Buskirk, Gisela Storz
bioRxiv 511675; doi: https://doi.org/10.1101/511675
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Identifying small proteins by ribosome profiling with stalled initiation complexes
Jeremy Weaver, Fuad Mohammad, Allen R. Buskirk, Gisela Storz
bioRxiv 511675; doi: https://doi.org/10.1101/511675

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