Cellular function of the GndA small open reading frame-encoded polypeptide during heat shock

Abstract

AbstractOver the past 15 years, hundreds of previously undiscovered bacterial small open reading frame (sORF)-encoded polypeptides (SEPs) of fewer than fifty amino acids have been identified, and biological functions have been ascribed to an increasing number of SEPs from intergenic regions and small RNAs. However, despite numbering in the dozens inEscherichia coli, and hundreds to thousands in humans, same-strand nested sORFs that overlap protein coding genes in alternative reading frames remain understudied. In order to provide insight into this enigmatic class of unannotated genes, we characterized GndA, a 36-amino acid, heat shock-regulated SEP encoded within the +2 reading frame of thegndgene inE. coliK-12 MG1655. We show that GndA pulls down components of respiratory complex I (RCI) and is required for proper localization of a RCI subunit during heat shock. At high temperature GndA deletion (ΔGndA) cells exhibit perturbations in cell growth, NADH+/NAD ratio, and expression of a number of genes including several associated with oxidative stress. These findings suggest that GndA may function in maintenance of homeostasis during heat shock. Characterization of GndA therefore supports the nascent but growing consensus that functional, overlapping genes occur in genomes from viruses to humans.Significance StatementSame-strand overlapping, or nested, protein coding sequences optimize the information content of size-constrained viral genomes, but were previously omitted from prokaryotic and eukaryotic genome annotations. It was therefore surprising when dozens of nested sORFs were recently discovered in bacteria. Our case study ofE. coliGndA supports the hypothesis that overlapping genes may exist because they encode proteins with related functions. More broadly, characterization of nested sORFs may revise our understanding of the architecture of bacterial and eukaryotic genes.