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 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 ribosoma subunits, tRNAMet, 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.
McNicholas
P
P
Department of Molecular Biology and Biochemistry, Wesleyan University, Middletown, CT 06459, USA.
Salavati
R
R
Oliver
D
D
D000251:Adenosine Triphosphatases / Q000235:genetics*; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics*; D001482:Base Composition; D001483:Base Sequence; D004926:Escherichia coli / Q000235:genetics*; D029968:Escherichia coli Proteins; D016368:Frameshift Mutation; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial; D026901:Membrane Transport Proteins; D008957:Models, Genetic; D008969:Molecular Sequence Data; D016296:Mutagenesis; D016297:Mutagenesis, Site-Directed; D009690:Nucleic Acid Conformation; D017354:Point Mutation; D014176:Protein Biosynthesis; D012329:RNA, Bacterial / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D012333:RNA, Messenger / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D012045:Regulatory Sequences, Nucleic Acid; D012270:Ribosomes / Q000378:metabolism; D000069816:SEC Translocation Channels; D000081416:SecA Proteins
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D026901:Membrane Transport Proteins; D012329:RNA, Bacterial; D012333:RNA, Messenger; D000069816:SEC Translocation Channels; D000251:Adenosine Triphosphatases; D000081416:SecA Proteins