Transcriptional activation by FNR and CRP: reciprocity of binding-site recognition.
Anaerobic expression of the focA pfl operon is dependent on the transcription 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 impairs anaerobic expression of the complete operon. Mutations were introduced into this binding site to create a consensus recognition site for the cAMP-receptor protein, CRP (CC-site), and one that was recognised by both CRP and FNR (CF-site). Transcription directed by these mutant binding sites in vivo in different promoter constructs was analysed by primer extension and by constructing lacZ operon fusions. With a derivative including only the P6 promoter and the CF-binding site, transcription was shown to be independent of oxygen and was activated by CRP or FNR. In agreement with previous findings, FNR only activated transcription anaerobically. In a construct including the CC-binding site transcription was strong. CRP dependent and initiated at the identical site to the wild-type promoter. Transcription activation from the CC-site was exquisitely sensitive to low cAMP concentration. Surprisingly, in a crp mutant, anaerobically inducible, FNR-dependent transcription directed by the CC-site was detected, indicating that FNR can recognise a consensus CRP-binding site in vivo. A strain unable to synthesise CRP or FNR exhibited no transcription from the P6 promoter. Essentially the same results were observed in a series of constructs that also included the promoter P7 and its regulatory sequences. Evidence is also presented which demonstrates that CRP activates transcription from the natural FNR-binding site of the P6 promoter. In vitro DNA-binding studies showed that CRP specifically interacted with the FNR-binding site, protecting exactly the same sequence as that protected by the FNR protein. Interaction of CRP with the natural FNR-binding site was reduced greater than 50-fold compared to its interaction with the mutant CC-binding site. Although we could not demonstrate that FNR interacted 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 further support to the idea that a single functional DNA half-site is sufficient to direct binding and transcription activation by a dimeric transcription factor.
Sawers
G
G
Nitrogen Fixation Laboratory, John Innes Centre, Norwich, UK. gary.sawers@bbsrc
Kaiser
M
M
Sirko
A
A
Freundlich
M
M
D000332:Aerobiosis; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D002352:Carrier Proteins; D003001:Cloning, Molecular; D000242:Cyclic AMP / Q000378:metabolism; D002373:Cyclic AMP Receptor Protein / Q000235:genetics / Q000378:metabolism*; D017931:DNA Primers / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial; D007506:Iron-Sulfur Proteins / Q000235:genetics / Q000378:metabolism*; D007763:Lac Operon; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000235:genetics; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D015533:Transcriptional Activation
D001426:Bacterial Proteins; D002352:Carrier Proteins; D002373:Cyclic AMP Receptor Protein; D017931:DNA Primers; D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; C060704:FNR protein, E coli; D007506:Iron-Sulfur Proteins; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D000242:Cyclic AMP