DUAL REGULATION OF ESCHERICHIA-COLI SECA TRANSLATION BY DISTINCT UPSTREAM ELEMENTS

The regulation of the Escherichia coli secA gene, whose translation is auto-repressed except when protein secretion becomes limiting, was in vestigated using a combination of genetic and biochemical approaches. Oligonucleotide-directed deletion and point mutagenesis was used to sh ow that only the last quarter of the upstream gene, geneX, and the gen eX-secA intergenic are essential for proper regulation. This region pr eviously shown to contain a secretion-responsive element contains two predicted helices, helix I and II, the latter of which would occlude t he secA Shine-Dalgarno sequence. Mutations that destabilized the lower portion of helix II increased secA basal expression, reduced auto-rep ression 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 b ase-pairing restored secA regulation to wild-type levels or nearly so, suggesting that this helix does play a role in secA auto-regulation i n vivo. In contrast, mutations in the lower portion of helix I decreas ed SECA basal expression, reduced auto-repression by SecA protein, and abolished the responsiveness of secA expression to a protein export b lock. In this latter case introduction of compensatory mutations into helix I that were predicted to restore base-pairing did not restore pr oper secA regulation, indicating that specific nucleotides in this reg ion are required for normal secA regulation. Primer-extension inhibiti on (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 ribo some binding at the secA translation initiation site, while mutations that perturbed helix I decreased ribosome binding at this site. Our re sults suggest strongly that there is a system of dual regulation of se cA translation, whereby helix I serves as an activator element while h elix II serves as a repressor element. (C) 1997 Academic Press Limited