title: A novel indirect sequence readout component in the E. coli cyclic AMP receptor protein operator.
abstract: The cyclic AMP receptor protein (CRP) from Escherichia coli has been extensively studied for several decades. In particular, a detailed characterization of CRP interaction with DNA has been obtained. The CRP dimer recognizes a consensus sequence AANTGTGANNNNNNTCACANTT through direct amino acid nucleobase interactions in the major groove of the two operator half-sites. Crystal structure analyses have revealed that the interaction results in two strong kinks at the TG/CA steps closest to the 6-base-pair spacer (N6). This spacer exhibits high sequence variability among the more than 100 natural binding sites in the E. coli genome, but the exact role of the N6 region in CRP interaction has not previously been systematic examined. Here we employ an in vitro selection system based on a randomized N6 spacer region to demonstrate that CRP binding to the lacP1 site may be enhanced up to 14-fold or abolished by varying the N6 spacer sequences. Furthermore, on the basis of sequence analysis and uranyl (UO2(2+)) probing data, we propose that the underlying mechanism relies on N6 deformability.
authors.affiliation: Department of Cellular and Molecular Medicine, Panum Institute, University of Copenhagen , Blegdamsvej 3, DK-2200 Copenhagen N, Denmark.
authors.forename: Søren
authors.initials: S
authors.lastname: Lindemose
authors.forename: Peter Eigil
authors.initials: PE
authors.lastname: Nielsen
authors.forename: Poul
authors.initials: P
authors.lastname: Valentin-Hansen
authors.forename: Niels Erik
authors.initials: NE
authors.lastname: Møllegaard
mesh_terms: D001483:Base Sequence; D001665:Binding Sites; D016384:Consensus Sequence; D018983:DNA Footprinting; D004269:DNA, Bacterial / Q000737:chemistry* / Q000235:genetics; D021901:DNA, Intergenic / Q000737:chemistry* / Q000235:genetics; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics; D016680:Genome, Bacterial; D007763:Lac Operon / Q000235:genetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011485:Protein Binding; D011953:Receptors, Cyclic AMP / Q000737:chemistry* / Q000235:genetics
chemical_list: D004269:DNA, Bacterial; D021901:DNA, Intergenic; D004268:DNA-Binding Proteins; D029968:Escherichia coli Proteins; D011953:Receptors, Cyclic AMP; C047947:cyclic AMP receptor protein, E coli