Mode of DNA binding by SopA protein of Escherichia coli F plasmid

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Abstract
The binding of SopA to the promoter region of its own gene, in which four copies of SopA’s recognition sequence, 5′-CTTTGC-3′, are arrayed asymmetrically, was examined in vitro. Titration using electrophoretic mobility shift assay showed that the stoichiometry of SopA protomers to the promoter-region DNA is 4 and that the binding is highly co-operative. The co-operativity was corroborated by EMSA and DNase I footprinting for a number of mutant DNA fragments in which 5′-CTTTGC-3′ was changed to 5′-CTTACG-3′. EMSA in the style of circular permutation showed that SopA bends DNA. Mutation at either outermost binding site had a different effect on DNA bending by SopA, reflecting the asymmetry in the arrangement of the binding sites, for which the results of DNase I footprinting were in agreement. Gel filtration chromatography and analytical ultracentrifugation of free SopA showed that the protein can exist as a monomer and oligomers in the absence of ATP. Hence, the results indicate that the co-operativity in SopA’s DNA binding is based on its intrinsic protein–protein interaction modified by DNA interaction.

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