Rescue of Escherichia coli auxotrophy by de novo small proteins

Abstract

Increasing numbers of small proteins with diverse physiological roles are being identified and characterized in both prokaryotic and eukaryotic systems, but the origins and evolution of these proteins remain unclear. Recent genomic sequence analyses in several organisms suggest that new functions encoded by small open reading frames (sORFs) may emerge de novo from noncoding sequences. However, experimental data demonstrating if and how randomly generated sORFs can confer beneficial effects to cells are limited. Here we show that by up-regulating hisB expression, de novo small proteins (≤ 50 amino acids in length) selected from random sequence libraries can rescue Escherichia coli cells that lack the conditionally essential SerB enzyme. The recovered small proteins are hydrophobic and confer their rescue effect by binding to the 5' end regulatory region of the his operon mRNA, suggesting that protein binding promotes structural rearrangements of the RNA that allow increased hisB expression. This study adds RNA regulatory elements as another interacting partner for de novo proteins isolated from random sequence libraries, and provides further experimental evidence that small proteins with selective benefits can originate from the expression of nonfunctional sequences.

Random, de novo, and conserved proteins: How structure and disorder predictors perform differently

“…However, to which extent is yet unknown, [8][9][10] Several studies have previously utilized randomly generated proteins to draw conclusions about the origin, structure, and activities of de novo proteins. [11][12][13][14][15][16][17] De novo proteins, particularly those of a more recent origin, have been found to differ from evolutionary conserved proteins and instead exhibit a similarity to random proteins, as they have not yet been subjected to natural selection. 8,9,13 Libraries of random sequences constrained only by a close to natural distribution of amino acids have been shown to form secondary structural elements.…”

“…Devoid of sequence homology outside their evolutionary trajectory, de novo proteins are considered to be distant in sequence space from evolutionarily conserved proteins and might instead resemble unevolved random proteins. However, to which extent is yet unknown, 8–10 Several studies have previously utilized randomly generated proteins to draw conclusions about the origin, structure, and activities of de novo proteins 11–17 . De novo proteins, particularly those of a more recent origin, have been found to differ from evolutionary conserved proteins and instead exhibit a similarity to random proteins, as they have not yet been subjected to natural selection 8,9,13 .…”

Large-scale investigation of species-specific orphan genes in the human gut microbiome elucidates their evolutionary origins

“…Future studies that leverage the ever-increasing global metagenomic sequences will be useful to tackle that question. Additionally, experimental evidence shows that the emergence of functional proteins via de novo gene evolution from noncoding sequences can provide adaptation in Escherichia coli (Babina et al 2023; Frumkin & Laub 2023). Thus, de novo evolution may be more plausible, and prevalent, than usually assumed.…”

A Synergistic, Cultivator Model of De Novo Gene Origination

“…One particularly interesting class of studies tackles this evolvability issue directly by presupposing some selective biological function. A large library of randomly generated synthetic polypeptides is then screened for the ability of fulfill this function ( Neme et al 2017 ; Babina et al 2023 ; Frumkin and Laub 2023 ). These studies are thus able to control for putative function by screening for increased reproductive capacity ( Neme et al 2017 ), the deactivation of a known toxin ( MazF/ramF ) ( Frumkin and Laub 2023 ), or rescue of auxotrophy (Δ serB ) ( Babina et al 2023 ), thereby empirically demonstrating at least the possibility for the evolution of such proteins de novo.…”