Hayes F, Austin S
Laboratory of Chromosome Biology, NCI-Frederick Cancer Research and Development Center, MD 21702.
J Mol Biol. 1994 Oct 21;243(2):190-8. doi: 10.1006/jmbi.1994.1646.
The parS site of the P1 plasmid promotes active partition of P1 to daughter cells when the P1 ParA and ParB proteins are provided. The structure of parS was modified by substituting portions of the sequence with synthetic oligonucleotides and testing partition activity of the resulting mutants in an in vivo assay. The boundaries of the site were defined. They enclose a 74 bp region with a central integration host factor (IHF) binding region flanked by two arms containing heptamer and hexamer ParB binding motifs. The IHF binding region was shown to be important for partition activity but could be replaced by sequences containing A tracts that induce static bends in the DNA. The properties of sites with spacer sequences of different lengths inserted at one of five different locations led to the following conclusions. (1) The spacing between the heptamer and hexamer ParB binding motifs in both arms is critical for function. (2) Optimum partition activity requires that the parS site arms are bent toward each other with specific faces of the two helices facing each other. (3) Both arms show torsional rigidity in the active complex. (4) The left arm is laterally inflexible and activity is lost when it is extended unless the right arm is similarly extended. (5) The right arm is laterally flexible so that, when it is extended by an integral number of turns of the helix, it can still align properly with a left arm of wild-type length. The results suggest that right-arm flexibility is promoted by an A + T-rich region that is essential for IHF binding and lies adjacent to the IHF binding consensus motif. Inherent flexibility of this A + T-rich region also appears to account for the residual activity of parS sites in which the IHF binding consensus has been destroyed by multiple point mutations. The results are consistent with a proposed structure in which specific alignment of the parS site arms by an IHF-promoted bend allows them to be linked by bifunctional ParB protein binding. We suggest that such a structure might be involved in the specific pairing and unpairing of daughter plasmids during partition by an isomerization reaction.
当提供P1质粒的ParA和ParB蛋白时，P1质粒的parS位点可促进P1向子细胞的主动分配。通过用合成寡核苷酸替换部分序列并在体内试验中测试所得突变体的分配活性，对parS的结构进行了修饰。确定了该位点的边界。它们包围了一个74 bp的区域，该区域有一个位于中央的整合宿主因子（IHF）结合区，两侧是两条含有七聚体和六聚体ParB结合基序的臂。已证明IHF结合区对分配活性很重要，但可被含有能诱导DNA静态弯曲的A序列的序列所取代。在五个不同位置之一插入不同长度间隔序列的位点的特性得出了以下结论。（1）两条臂中七聚体和六聚体ParB结合基序之间的间距对功能至关重要。（2）最佳分配活性要求parS位点的臂彼此弯曲，两条螺旋的特定面相互面对。（3）在活性复合物中，两条臂均表现出扭转刚性。（4）左臂在横向是不可弯曲的，当它延伸时活性丧失，除非右臂也同样延伸。（5）右臂在横向是可弯曲的，因此，当它通过螺旋的整数圈延伸时，它仍然可以与野生型长度的左臂正确对齐。结果表明，右臂的柔韧性是由一个富含A+T的区域促进的，该区域对IHF结合至关重要，且位于IHF结合共有基序附近。这个富含A+T区域的固有柔韧性似乎也解释了parS位点的残余活性，在这些位点中，IHF结合共有序列已被多个点突变破坏。这些结果与一种 proposed 结构一致，在该结构中，由IHF促进的弯曲使parS位点的臂进行特定排列，从而使它们能够通过双功能ParB蛋白结合而连接。我们认为，这样的结构可能参与了分配过程中通过异构化反应使子代质粒进行特定配对和解配对。 （注：原文中“proposed”未翻译，可能是拼写错误，推测为“proposed”，意为“提出的”）
