THE 60 NUCLEOTIDE OCCR OPERATOR CONTAINS A SUBSITE ESSENTIAL AND SUFFICIENT FOR OCCR BINDING AND A 2ND SUBSITE REQUIRED FOR LIGAND-RESPONSIVE DNA BENDING

OccR is a transcriptional regulatory protein of Agrobacterium tumefaci ens that activates the occQ operon in response to octopine, an arginin e derivative released from plant tumors. OccR binds to its operator wi th similar affinity and the same stoichiometry in the presence or abse nce of octopine, but octopine shortens the protein's DNase I footprint and partially relaxes an OccR-incited DNA bend. In this study resecti ons and other alterations of the operator were used to demonstrate tha t 19 nucleotides near the end of the operator furthest from the occa p romoter were essential for high affinity OccR binding. This sequence, denoted the high affinity subsite, was sufficient for binding, provide d that the deleted operator sequences were replaced with vector-derive d DNA. The same number of OccR monomers bound to resected operators as to the wild-type operator, and OccR Mras able to protect vector-deriv ed sequences adjacent to the high affinity subsite. Sequences at the p romoter proximal end of the operator were required for wild-type patte rns 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 f ully or partially locked high angle bends. This suggests that the prom oter proximal half of the operator may contain at least two sites requ ired for wild-type ligand-responsive DNA bending. These mutations also provided evidence that the partial relaxation of this bend by octopin e may be essential for occQ activation. (C) 1995 Academic Press Limite d