title: Redox-dependent DNA distortion in a SoxR protein-promoter complex studied using fluorescent probes.
abstract: The [2Fe-2S] transcriptional factor SoxR, a member of the MerR family, is regulated by the reversible oxidation and reduction of [2Fe-2S] clusters and functions as a sensor of oxidative stress in Escherichia coli. In the oxidized state, distortion of the target DNA promoter region initiates transcription by RNA polymerase, thereby activating transcription. The inactive reduced state of the protein has remained uncharacterized. Here, we directly observed redox-dependent conformational changes in the promoter DNA by site-specifically replacing selected adenine (A) and cytosine (C) bases in the promoter oligonucleotide with the fluorescent probes 2-aminopurine (2Ap) and pyrrolocytosine (pyrrolo-dC), respectively. Reduction of the [2Fe-2S] cluster in the SoxR-DNA complex dramatically weakened the fluorescence intensity of the 2Ap moieties incorporated into the central part of the DNA. In contrast, the fluorescence of 2Ap moieties incorporated at A in other regions and the fluorescence of pyrrolo-dC moieties in the central region of the DNA (C3 and C3') were only slightly decreased by the reduction. These results strongly suggest that the redox change causes a large conformational change within a region confined to the central A-T base pairs in the promoter region of the DNA.
authors.affiliation: The Institute of Scientific and Industrial Research, Osaka University, Mihogaoka 8-1, Ibaraki, Osaka 567-0047, Japan.
authors.forename: Mayu
authors.initials: M
authors.lastname: Fujikawa
authors.affiliation: The Institute of Scientific and Industrial Research, Osaka University, Mihogaoka 8-1, Ibaraki, Osaka 567-0047, Japan kobayasi@sanken.osaka-u.ac.jp.
authors.forename: Kazuo
authors.initials: K
authors.lastname: Kobayashi
authors.affiliation: The Institute of Scientific and Industrial Research, Osaka University, Mihogaoka 8-1, Ibaraki, Osaka 567-0047, Japan.
authors.forename: Takahiro
authors.initials: T
authors.lastname: Kozawa
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D004247:DNA / Q000737:chemistry*; D005456:Fluorescent Dyes / Q000378:metabolism*; D010084:Oxidation-Reduction; D011401:Promoter Regions, Genetic; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D005456:Fluorescent Dyes; D014157:Transcription Factors; C070724:SoxR protein, Bacteria; D004247:DNA
keywords: DNA-protein complex; fluorescence; iron-sulfur cluster; redox regulation; transcription factor