Dual regulation of Escherichia coli secA translation by distinct upstream elements

Abstract

The regulation of the Escherichia coli secA gene, whose translation is auto-repressed except when protein secretion becomes limiting, was investigated using a combination of genetic and biochemical approaches. Oligonucleotide-directed deletion and point mutagenesis was used to show that only the last quarter of the upstream gene, geneX, and the geneX- secA intergenic are essential for proper regulation. This region previously shown to contain a secretion-responsive element contains two predicted helices, helix I and II, the latter of which would occlude the secA Shine-Dalgarno sequence. Mutations that destabilized the lower portion of helix II increased secA basal expression, reduced auto-repression by SecA protein, but retained a normal pattern of derepression of secA expression during a protein export block. The introduction of compensatory mutations into helix II that were predicted to restore base-pairing restored secA regulation to wild-type levels or nearly so, suggesting that this helix does play a role in secA auto-regulation in vivo. In contrast, mutations in the lower portion of helix I decreased secA basal expression, reduced auto-repression by SecA protein, and abolished the responsiveness of secA expression to a protein export block. In this latter case introduction of compensatory mutations into helix I that were predicted to restore base-pairing did not restore proper secA regulation, indicating that specific nucleotides in this region are required for normal secA regulation. Primer- extension inhibition (toeprint) analysis with 30 S ribosomal subunits, tRNA f Met , and a model segment of geneX- secA RNA carrying the relevant mutations was used to show that mutations that destabilized helix II increased ribosome binding at the secA translation initiation site, while mutations that perturbed helix I decreased ribosome binding at this site. Our results suggest strongly that there is a system of dual regulation of secA translation, whereby helix I serves as an activator element while helix II serves as a repressor element.