Mutations in the occQ operator that decrease OccR-induced DNA bending do not cause constitutive promoter activity.
OccR is a LysR-type transcriptional regulator of Agrobacterium tumefaciens that positively regulates the octopine catabolism operon of the Ti plasmid. Positive control of the occ genes occurs in response to octopine, a metabolite released from plant tumors. Octopine causes DNA-bound OccR to undergo a conformational change from an inactive to an active state; this change is marked by a decrease in footprint length from 55 to 45 nucleotides as well as a relaxation of a high angle DNA bend. In this study, we first used gel filtration chromatography to show that OccR is dimeric in solution, and we used gel shift assays to show that OccR is tetrameric when bound to DNA. We then created a series of site-directed mutations in the OccR-binding site. Some mutations were designed to lock OccR-DNA complexes into a conformation resembling the inactive conformation, whereas other mutations were designed to lock complexes into the active conformation. These mutations altered the conformation of OccR-DNA complexes and their responses to octopine in ways that we had predicted. As expected, operator mutations that locked complexes into a conformation having a long footprint and a high angle DNA bend blocked activation by octopine in vivo. Surprisingly, however, mutations that lock OccR into a short footprint and low angle DNA bend failed to cause the protein to function constitutively. Furthermore, some of the latter mutations interfered with activation by octopine. We conclude that locking OccR into a conformation having a short footprint is not sufficient to cause constitutive activation, and octopine must cause at least one additional conformational change in the protein.
Akakura
Reiko
R
Department of Microbiology, Cornell University, Ithaca, New York 14853, USA.
Winans
Stephen C
SC
D016960:Agrobacterium tumefaciens / Q000235:genetics*; D001120:Arginine / Q000031:analogs & derivatives* / Q000378:metabolism*; D001426:Bacterial Proteins / Q000235:genetics*; D001483:Base Sequence; D002850:Chromatography, Gel; D018983:DNA Footprinting; D017931:DNA Primers; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000235:genetics; D019281:Dimerization; D015964:Gene Expression Regulation, Bacterial; D005838:Genotype; D007700:Kinetics; D008969:Molecular Sequence Data; D009876:Operon; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D015183:Restriction Mapping; D014157:Transcription Factors / Q000235:genetics*
D001426:Bacterial Proteins; D017931:DNA Primers; D004269:DNA, Bacterial; D004268:DNA-Binding Proteins; D014157:Transcription Factors; C073469:OccR protein, Bacteria; C005394:octopine; D001120:Arginine