Key: Click on "details" for more information on a particular experiment or article. "Result" is a short description of the experiment result. "Methods" use a ontology to describe the methods of the experiment.
  Results:Regulatory site mappingMethods:Gel retardation; Mutant analysis; Sequence analysis
Mobility shift assay experiments were used to confirm that Cra (FruR) binds to the nir promoter and that the observed Cra-dependent repression of nir expression is not due indirect effects. In these experiments, the nir promoter in plasmid and crude protein extracts from Cra- and Cra+ strains were used. No retardation of the nir promoter fragment was observed with Cra # extracts. However, with Cra+ extracts, a single species with retarded electrophoretic mobility was observed. Furthermore, the amount of retarded nir promoter fragment decreased when 0.5 mM fructose 1-phosphate was included in the reaction, confirming that the observed retardation is due to the binding of Cra. Within the nir promoter sequence, from #23 bp to #10 bp with respect to the transcription start, there is an im perfect palindrome, (GC)TGAATCGTTAAG, that is identical to the proposed consensus for Cra binding sites, (GC)TGAAACGTTTCA, at 10 out of 14 positions As in the case of other Cra binding sites, the upstream half of the palindrome corresponds better to the consensus than the downstream half, which, at the nir promoter, overlaps the #10 hexamer and, thus, must be subject to additional sequence constraints. In addition, the putative Cra site at the nir promoter also contains the GC motif, located immediately upstream of the inverted repeat, that has been shown to be of prime importance for high-affinity Cra binding in vitro.
Experiment Elements
site: FruR retarded region at nir operon Escherichia coli
site: putative FruR site at nir operon Escherichia coli
regulator: FruR Escherichia coli
operon: nir Escherichia coli
effector: Fructose-1-phosphate
Tyson K,Busby S,Cole J
Catabolite regulation of two Escherichia coli operons encoding nitrite reductases: role of the Cra protein.Arch Microbiol 1997 Sep;168(3):240-4. (details)
PubMed: 9382703