Identifying small proteins by ribosome profiling with stalled initiation complexes

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 these regulators remains unknown because conventional annotation pipelines usually exclude such 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 selected 41 smORFs for chromosomal tagging and detected small protein synthesis for all but 3. 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 translation of larger downstream genes. These results demonstrate the utility of this method for identifying new genes, regardless of their genomic context. Ribosome profiling of E. coli MG1655 cultures treated with Onc112, an antibiotic that traps ribosomes at start codons, as a means to identify start sites in intergenic regions.