Direct evidence for mRNA binding and post-transcriptional regulation by Escherichia coli aconitases

Escherichia coil contains a stationary-phase aconitase (AcnA) that is induc ed by Biomolecular Research, iron and oxidative stress, and a major but les s stable aconitase (AcnB) Department of Molecular synthesized during expone ntial growth, These enzymes were shown to resemble the bifunctional iron-re gulatory proteins (IRP1)/cytoplasmic aconitases of vertebrates in having al ternative mRNA-binding and catalytic activities. Affinity chromatography an d gel retardation analysis showed that the AcnA and AcnB ape-proteins each interact with the 3' untranslated regions (3'UTRs) of acnA and acnB mRNA at physiologically significant protein concentrations, AcnA and AcnB synthesi s was enhanced in vitro by the apo-aconitases and this enhancement was abol ished by 3'UTR deletion from the DNA templates, presumably by loss of acn-m RNA stabilization by bound apo-aconitase, In vivo studies showed that altho ugh total aconitase activity is lowered during oxidative stress, synthesis of the AcnA and AcnB proteins and the stabilities of acnA and acnB mRNAs bo th increase, suggesting that inactive aconitase mediates a post-transcripti onal positive autoregulatory switch. Evidence for an iron-sulphur-cluster-d ependent switch was inferred from the more than threefold higher mRNA-bindi ng affinities of the apo-aconitases relative to the holo-enzymes. Thus by m odulating translation via site-specific interactions between ape-enzyme and relevant transcripts, the aconitases provide a new and rapidly reacting co mponent of the bacterial oxidative stress response.