Mohsen, J. J.; Mohsen, M. G.; Jiang, K.; Landajuela, A.; Quinto, L.; Isaacs, F. J.; Karatekin, E.; Slavoff, S. A.

Over 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 in Escherichia 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 the gnd gene in E. coli K-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 ({Delta}GndA) 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.