Cellular function of the GndA microprotein during heat shock
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Over the past 15 years, hundreds of previously undiscovered bacterial small open reading frames (sORFs) encoding microproteins of fewer than fifty amino acids have been identified. Biological functions have been ascribed to an increasing number of microproteins from intergenic regions and small RNAs, and many play integral roles in bacterial stress responses. However, despite numbering in the dozens in Escherichia coli , and hundreds in humans, same-strand frameshifted sORFs that internally overlap protein coding sequences remain understudied. To provide insight into nested genes, we characterized GndA, a frameshifted 36-amino acid microprotein nested within the 6-phosphogluconate dehydrogenase (6PGD) coding sequence. Using precise genome editing, we demonstrate independent contributions of GndA and 6PGD to cell growth at high temperature. GndA associates with membrane-associated complexes associated with electron transport and ATP generation, and supports ATP homeostasis during heat shock. Functional characterization of GndA thus adds to the catalog of bacterial microproteins that function in stress responses, while providing clear genetic evidence for the importance of an overlapping gene to cellular fitness.
Significance Statement
Same-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 numerous nested sORFs were recently discovered in bacteria and eukaryotes. Our case study of E. coli GndA supports the emerging model that overlapping genes can be expressed and contribute to cellular fitness, as GndA expression supports cell growth at elevated temperature. Characterization of nested sORFs may therefore revise our understanding of the complexity of bacterial and eukaryotic genes.
