Received JRevised and Accepted September 12, 2000. Thus, the RegR box may assist in the identification of new RegR target genes not only in B.japonicum but also in other α-proteobacteria possessing RegR-like response regulators. A comparison of the RegR box with functional binding sites of the RegR-like regulator RegA of Rhodobacter capsulatus revealed considerable similarities. Based on these results, we defined a DNA motif comprising those nucleotides that are critical for RegR binding (RegR box 5′-GNG A GC A GTTNNGNCGC-3′). Notably, all 11 critical nucleotides were located either within the half sites of the inverted repeat (four nucleotides in each half site) or in the 5 bp spacer that separates the half sites (three nucleotides). This led to the identification of 11 critical nucleotides within a 17 bp minimal RegR binding site centered at position –64 upstream of the fixR-nifA transcription start site. In a parallel approach, band-shift experiments were performed with oligonucleotides comprising defined point or deletion mutations in the fixR UAS. The selected sequences comprised an imperfect inverted repeat (GCGGC-N 5-GTCGC) which is highly similar to an imperfect inverted repeat in the fixR UAS (GCGAC-N 5-GACGC). Here, we used an in vitro binding-site selection assay (SELEX) to more precisely define the DNA-binding specificity of RegR. In previous in vivo studies, we identified a 32 bp upstream activating sequence located around position –68, which is essential for RegR-dependent expression of the fixR-nifA operon. The only target known so far is the fixR-nifA operon, encoding the redox-responsive transcription factor NifA, which activates many genes required for symbiotic nitrogen fixation in soybean nodules.
RegR is the response regulator of the RegSR two-component regulatory system in Bradyrhizobium japonicum.
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