Definition of a consensus DNA-binding site for the Escherichia coli pleiotropic regulatory protein, FruR.
The FruR regulator of Escherichia coli controls the initiation of transcription of several operons encoding a variety of proteins involved in carbon and energy metabolism. The sequence determinants of the FruR-binding site were analysed by using 6x His-tagged FruR and a series of double-stranded randomized oligonucleotides. FruR consensus binding sites were selected and characterized by several consecutive rounds of the polymerase chain reaction-assisted binding-site selection method (BSS) using nitrocellulose-immobilized DNA-binding protein. FruR was demonstrated to require, for binding, an 8 bp left half-site motif and a 3 bp conserved right half-site with the following sequence: 5'-GNNGAATC/GNT-3'. In this sequence, the left half-site AATC/ consensus tetranucleotide is a typical motif of the DNA-binding site of the regulators of the GalR-Lacl family. On the other hand, the high degree of degeneracy found in the right half-site of this palindrome-like structure indicated that FruR, which is a tetramer in solution, interacts asymmetrically with the two half-sites of its operator. However, potentially FruR-target sites showing a high degree of symmetry were detected in 13 genes/operons. Among these, we have focused our interest on the pfkA gene, encoding phosphofructo-kinase-1, which is negatively regulated by FruR.
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Institut de Biologie et Chimie des Protéines, Centre National de la Recherche Scientifique, Lyon, France.
Bonod-Bidaud
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Geourjon
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Deléage
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Cozzone
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Cortay
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D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D016384:Consensus Sequence; D018983:DNA Footprinting; D019175:DNA Methylation; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D003850:Deoxyribonuclease I; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism*; D029968:Escherichia coli Proteins; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D009876:Operon; D010732:Phosphofructokinase-1 / Q000235:genetics; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D012689:Sequence Homology, Nucleic Acid; D056968:Lac Repressors
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D003850:Deoxyribonuclease I; D029968:Escherichia coli Proteins; D010732:Phosphofructokinase-1; D012097:Repressor Proteins; C489416:FruR protein, E coli; C068837:FruR protein, Bacteria; D056968:Lac Repressors