TRANSCRIPTIONAL ACTIVATION BY FNR AND CRP - RECIPROCITY OF BINDING-SITE RECOGNITION

Anaerobic expression of the focA pfl operon is dependent on the transc ription factors ArcA and FNR and transcription is directed by multiple , anaerobically regulated promoters. A FNR-binding site is centred at -41.5 bp relative to the P6 promoter, inactivation of which severely i mpairs anaerobic expression of the complete operon. Mutations were int roduced into this binding site to create a consensus recognition site for the cAMP-receptor protein, CRP (CC-site), and one that was recogni sed by both CRP and FNR (CF-site). Transcription directed by these mut ant binding sites in vivo in different promoter constructs was analyse d by primer extension and by constructing lacZ operon fusions. With a derivative including only the P6 promoter and the CF-binding site, tra nscription was shown to be independent of oxygen and was activated by CRP or FNR. In agreement with previous findings, FNR only activated tr anscription anaerobically. In a construct including the CC-binding sit e transcription was strong, CRP dependent and initiated at the identic al site to the wild-type promoter. Transcription activation from the C C-site was exquisitely sensitive to low cAMP concentration. Surprising ly, in a crp mutant, anaerobically inducible, FNR-dependent transcript ion directed by the CC-site was detected, indicating that FNR can reco gnise a consensus CRP-binding site in vivo. A strain unable to synthes ise CRP or FNR exhibited no transcription from the P6 promoter. Essent ially the same results were observed in a series of constructs that al so included the promoter P7 and its regulatory sequences. Evidence is also presented which demonstrates that CRP activates transcription fro m the natural FNR-binding site of the P6 promoter. In vitro DNA-bindin g studies showed that CRP specifically interacted with the FNR-binding site, protecting exactly the same sequence as that protected by the F NR protein. Interaction of CRP with the natural FNR-binding site was r educed greater than 50-fold compared to its interaction with the mutan t CC-binding site. Although we could not demonstrate that FNR interact ed with the CC-binding site in vitro it did bind to the CF-site giving the same protection as observed with the wild-type FNR-binding site. FNR also activated transcription from the CF-site in vitro, giving fur ther support to the idea that a single functional DNA half-site is suf ficient to direct binding and transcription activation by a dimeric tr anscription factor.