Format

Send to

Choose Destination
MBio. 2019 Mar 5;10(2). pii: e02819-18. doi: 10.1128/mBio.02819-18.

Identifying Small Proteins by Ribosome Profiling with Stalled Initiation Complexes.

Author information

1
Division of Molecular and Cellular Biology, Eunice Kennedy Shriver National Institute of Child Health and Human Development, Bethesda, Maryland, USA.
2
Department of Molecular Biology and Genetics, Johns Hopkins School of Medicine, Baltimore, Maryland, USA.
3
Department of Molecular Biology and Genetics, Johns Hopkins School of Medicine, Baltimore, Maryland, USA buskirk@jhmi.edu storzg@mail.nih.gov.
4
Division of Molecular and Cellular Biology, Eunice Kennedy Shriver National Institute of Child Health and Human Development, Bethesda, Maryland, USA buskirk@jhmi.edu storzg@mail.nih.gov.

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 total number 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. Not only are the corresponding genes intergenic but they 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 functions 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.

KEYWORDS:

Ribo-seq; alternate ORFs; antisense; genome annotation; leader peptide; small protein

PMID:
30837344
PMCID:
PMC6401488
DOI:
10.1128/mBio.02819-18
[Indexed for MEDLINE]
Free PMC Article

Supplemental Content

Full text links

Icon for HighWire Icon for PubMed Central
Loading ...
Support Center