Altered-function mutations in the Agrobacterium tumefaciens OccR protein and in an OccR-regulated promoter

Abstract

OccR is a LysR-type transcriptional activator that controls the occQ and traR promoters of octopine-type Ti plasmids. The opine octopine converts OccR from a repressor to an activator of occQ, shortens the protein's DNase I footprint, and decreases the angle of an OccR-caused DNA bend at the occQ promoter. In this study we first localized the cis-acting DNA sequences required for regulated expression of occQ. To understand better the mechanism of activation of OccR, we isolated mutations both in the occQ promoter and in the occR gene which function differently from the wild type. An occQ promoter mutation that changes the putative -35 region of occQ from TTGACC to TTGACA increases the basal expression of occQ about 15-fold. Three mutations in occR were also identified, one of which activates occQ at fully constitutive levels in both the absence and presence of octopine. This mutation (E23G) is located in the first helix of a putative helix-turn-helix DNA-binding motif. The other two occR mutations cause the protein to detect much lower concentrations of octopine than wild-type OccR protein does. These mutations (F113L and G148D) are located in a region of the protein that is predicted to contain the ligand-binding site.