Genomes contain orders of magnitude more open reading frames (ORFs) than known protein coding genes, and recent work suggests there may be unannotated proteins present in even the best studied organisms. To address this gap, we used a high throughput reverse genetic toolkit to construct precise C-terminal fusions of a reporter (and control) to >120,000 ORFs in model E. coli . We found hundreds of unannotated significant hits, and individually detected >50 novel polypeptides by western blot, including ORFs within tRNA loci. Many ORFs overlap annotated genes in the sense orientation, and we found these are likely chimeric polypeptides produced by ribosomal frameshifting. Using degron based knockdowns, we identified unannotated proteins that have putative fitness effects, and we found a novel small protein that displays phenotypes consistent with a role in the mRNA degradosome. The observation of a range of unannotated translation products should lead to better annotation and understanding of the bacterial domain of life and motivates the continued exploration of genomes broadly. ### Competing Interest Statement The authors have declared no competing interest.

Graphics processing unit-accelerated MMseqs2 offers tremendous speedups for homology retrieval from metagenomic databases, query-centered multiple sequence alignment generation for structure predictio...

Promoters are DNA sequences that help to initiate transcription. Point mutations can create de-novo promoters, which can consequently transcribe inactive genes or create novel transcripts. We know lit...

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Structurally complex regions of the genome are increasingly recognized as engines of evolutionary convergence due to their propensity to generate recurrent gene duplications that give rise to similar ...

Nature Ecology & Evolution - A combination of computational methods applied to single-cell RNA sequencing data and genetic experiments shows that a small number of transcription factors are...

PhD student in Biochemistry, Disciplinary Domain of Science and Technology, Faculty of Chemistry, Department of Chemistry - BMC, Uppsala University