The sixty nucleotide OccR operator contains a subsite essential and sufficient for OccR binding and a second subsite required for ligand-responsive DNA bending.
OccR is a transcriptional regulatory protein of Agrobacterium tumefaciens that activates the occQ operon in response to octopine, an arginine derivative released from plant tumors. OccR binds to its operator with similar affinity and the same stoichiometry in the presence or absence of octopine, but octopine shortens the protein's DNase I footprint and partially relaxes an OccR-incited DNA bend. In this study, resections and other alterations of the operator were used to demonstrate that 19 nucleotides near the end of the operator furthest from the occQ promoter were essential for high affinity OccR binding. This sequence, denoted the high affinity subsite, was sufficient for binding, provided that the deleted operator sequences were replaced with vector-derived DNA. The same number of OccR monomers bound to resected operators as to the wild-type operator, and OccR was able to protect vector-derived sequences adjacent to the high affinity subsite. Sequences at the promoter proximal end of the operator were required for wild-type patterns of ligand-responsive DNA bending. A sequence alteration at the end furthest from the high affinity subsite caused a partially locked low angle DNA bend, while two more centrally localized mutations caused fully or partially locked high angle bends. This suggests that the promoter proximal half of the operator may contain at least two sites required for wild-type ligand-responsive DNA bending. These mutations also provided evidence that the partial relaxation of this bend by octopine may be essential for occQ activation.
D016960:Agrobacterium tumefaciens / Q000235:genetics / Q000378:metabolism; D001120:Arginine / Q000031:analogs & derivatives / Q000378:metabolism; D001426:Bacterial Proteins / Q000378:metabolism*; D001483:Base Sequence; D004247:DNA / Q000378:metabolism*; D018983:DNA Footprinting; D004269:DNA, Bacterial / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins; D008024:Ligands; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009875:Operator Regions, Genetic; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D014157:Transcription Factors / Q000378:metabolism*
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D008024:Ligands; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; C073469:OccR protein, Bacteria; C005394:octopine; D004247:DNA; D001120:Arginine