Mori H, Mori Y, Ichinose C, Niki H, Ogura T, Kato A, Hiraga S
Department of Molecular Genetics, Kumamoto University Medical School, Japan.
J Biol Chem. 1989 Sep 15;264(26):15535-41.
Mini-F plasmid has the trans-acting genes sopA and sopB and the cis-acting site sopC which are essential for accurate partitioning of plasmid DNA molecules into both daughter cells. In this study, we purified independently SopA and SopB proteins, analyzed the in vitro DNA-binding activity of these proteins by the gel retardation assay, and determined the precise binding sites of DNA by the footprinting method. SopA binds to four repeated sequences (CTTTGC) located in the promoter-operator region of the sopAB operon. The SopA binding activity is enhanced by the addition of SopB protein. SopB protein itself does not bind to this DNA region. These results suggest that the complex of SopA and SopB proteins autoregulate the expression of the sopA-sopB operon. On the other hand, SopB protein binds to the sopC region, in which 12 direct repeats of 43-base pairs nucleotides exist. SopB protein recognizes the inverted repeats of 7 base pairs in each direct repeats. SopA protein does not affect the SopB binding activity to the sopC DNA segment.
微小F质粒具有反式作用基因sopA和sopB以及顺式作用位点sopC，这些对于将质粒DNA分子准确分配到两个子细胞中至关重要。在本研究中，我们分别纯化了SopA和SopB蛋白，通过凝胶阻滞试验分析了这些蛋白的体外DNA结合活性，并通过足迹法确定了DNA的精确结合位点。SopA与位于sopAB操纵子启动子-操纵基因区域的四个重复序列（CTTTGC）结合。添加SopB蛋白可增强SopA的结合活性。SopB蛋白本身不与该DNA区域结合。这些结果表明，SopA和SopB蛋白复合物可自动调节sopA-sopB操纵子的表达。另一方面，SopB蛋白与sopC区域结合，该区域存在12个43个碱基对核苷酸的直接重复序列。SopB蛋白识别每个直接重复序列中7个碱基对的反向重复序列。SopA蛋白不影响SopB与sopC DNA片段的结合活性。
