Integration of three signals at the Escherichia coli nrf promoter: a role for Fis protein in catabolite repression.
Expression from the Escherichia coli nrf operon promoter is activated by the anaerobically triggered transcription factor, FNR, and by the nitrate/nitrite ion-controlled response regulators, NarL or NarP, but is repressed by the IHF and Fis proteins. Here, we present in vitro studies on the nrf promoter, using permanganate footprinting to measure open complex formation, and DNase I footprinting to monitor binding of the different regulators and the interactions between them. Our results show that open complex formation is completely dependent on FNR and is enhanced by NarL, but is repressed by IHF or Fis. NarL counteracts repression by IHF but is unable to alter repression by Fis. These results suggest mechanisms by which nrf promoter activity is modulated by the different factors. Expression from the nrf promoter is known to be repressed in rich media, especially in the presence of glucose, but the molecular basis of this is not understood. Here, we show that this catabolite repression is relieved by mutations that weaken the DNA site for Fis, improve the DNA site for FNR or improve the promoter -10 or -35 elements. Hence, Fis protein is a major factor responsible for catabolite repression at the nrf promoter, and Fis can override activation by FNR and NarL or NarP.
School of Biosciences, The University of Birmingham, Birmingham B15 2TT, UK.
Douglas F
DF
Browning
David C
DC
Grainger
Christine M
CM
Beatty
Alan J
AJ
Wolfe
Jeffrey A
JA
Cole
Stephen J W
SJ
Busby
D001483:Base Sequence; D018983:DNA Footprinting; D004269:DNA, Bacterial / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D024202:Electrophoretic Mobility Shift Assay; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000378:metabolism; D035021:Factor For Inversion Stimulation Protein; D015964:Gene Expression Regulation, Bacterial; D034982:Integration Host Factors / Q000378:metabolism; D007506:Iron-Sulfur Proteins / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D014157:Transcription Factors / Q000378:metabolism
D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; C060704:FNR protein, E coli; D035021:Factor For Inversion Stimulation Protein; C501034:Fis protein, E coli; D034982:Integration Host Factors; D007506:Iron-Sulfur Proteins; D014157:Transcription Factors; C440502:integration host factor, E coli; C081466:narP protein, E coli; C069780:NarL protein, E coli