PMID:9020977

Citation	McNicholas, P, Salavati, R and Oliver, D (1997) Dual regulation of Escherichia coli secA translation by distinct upstream elements. J. Mol. Biol. 265:128-41
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 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.
Links	PubMed Online version:10.1006/jmbi.1996.0723
Keywords	Adenosine Triphosphatases/genetics; Amino Acid Sequence; Bacterial Proteins/genetics; Base Composition; Base Sequence; Escherichia coli/genetics; Escherichia coli Proteins; Frameshift Mutation; Gene Expression Regulation, Bacterial; Genes, Bacterial; Membrane Transport Proteins; Models, Genetic; Molecular Sequence Data; Mutagenesis; Mutagenesis, Site-Directed; Nucleic Acid Conformation; Point Mutation; Protein Biosynthesis; RNA, Bacterial/chemistry; RNA, Bacterial/genetics; RNA, Bacterial/metabolism; RNA, Messenger/chemistry; RNA, Messenger/genetics; RNA, Messenger/metabolism; Regulatory Sequences, Nucleic Acid; Ribosomes/metabolism
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