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.

The advent of system-wide proteomic approaches has largely expanded the number of RNA-binding proteins (RBPs). This review discusses how recent discoveries are transforming our understanding of biolog...