Selection of a de novo gene that can promote survival of E. coli by modulating protein homeostasis pathways

Cells sometime adapt to challenging environments by turning non-functional loci into functional genes in a process termed de novo gene birth. But how proteins with random amino acid sequences integrate into existing cellular pathways to provide a benefit remains poorly understood. Here, we screened [~]108 random genes for their ability to rescue growth arrest of E. coli cells producing the ribonuclease toxin MazF. Approximately 2,000 random genes could promote growth by reducing transcription from the promoter driving mazF expression. Additionally, one gene, named random antitoxin of MazF (ramF), whose protein product was well-tolerated in cells and neutralized MazF by interacting with chaperones, leading to MazF proteolysis. We show that the specificity of RamF for MazF relative to other toxins relies on the degron-like function of MazFs first 10 amino acids. Finally, we demonstrate that random proteins can improve during evolution by identifying beneficial mutations that turned RamF into a more efficient inhibitor. Our work provides a mechanistic basis for how de novo gene birth can produce new, functional proteins that are integrated into complex cellular systems and provide a benefit to cells.