Funnell B E, Gagnier L
Department of Molecular and Medical Genetics, University of Toronto, Ontario, Canada.
J Biol Chem. 1993 Feb 15;268(5):3616-24.
The P1 plasmid prophage is partitioned by a very high affinity protein complex at its partition site, parS, that contains the P1 ParB protein and Escherichia coli integration host factor (IHF). ParB binds to regions of parS that flank the IHF binding site. In this report, we have examined the sequences to which ParB binds, the spatial relationship between them, and the effect of IHF on ParB binding patterns. Methylation protection and interference experiments were performed on supercoiled plasmids. Mutations that interfered with the action of both proteins in vivo were identified following random mutagenesis of parS. These studies revealed that ParB binds to a complicated, nonsymmetrical region in the right side of parS. ParB recognizes a partial copy of this sequence, TCGCCA, in the left side of parS with much lower affinity. The presence of IHF greatly facilitates the interaction of ParB with parS such that both sides bind with an equal affinity that is much greater than to either side alone. The stimulation by IHF is strongly influenced by helical phasing. These observations support the proposal that ParB is directed, by the bend created by IHF, to bind simultaneously to properly placed sequences flanking the IHF site.
P1质粒原噬菌体通过一种高亲和力的蛋白质复合物在其分区位点parS进行分区，parS包含P1 ParB蛋白和大肠杆菌整合宿主因子（IHF）。ParB与位于IHF结合位点两侧的parS区域结合。在本报告中，我们研究了ParB结合的序列、它们之间的空间关系以及IHF对ParB结合模式的影响。对超螺旋质粒进行了甲基化保护和干扰实验。在对parS进行随机诱变后，鉴定出了在体内干扰这两种蛋白质作用的突变。这些研究表明，ParB与parS右侧一个复杂的非对称区域结合。ParB在parS左侧识别该序列的部分拷贝TCGCCA，但其亲和力要低得多。IHF的存在极大地促进了ParB与parS的相互作用，使得两侧以相等的亲和力结合，且该亲和力远大于单独与任一侧结合的亲和力。IHF的刺激受到螺旋相位的强烈影响。这些观察结果支持了这样一种观点，即ParB受IHF产生的弯曲引导，同时与位于IHF位点两侧合适位置的序列结合。
