Koblan K S, Ackers G K
Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, Missouri 63110.
Biochemistry. 1991 Aug 6;30(31):7822-7. doi: 10.1021/bi00245a023.
The effects of monovalent salt activity on the site-specific and cooperative interactions of cI repressor with its three operator sites OR were studied by using quantitative DNase I footprint titration methods. Individual-site binding isotherms were obtained for binding repressor dimers to each site of wild-type OR and to mutant operator templates in which binding to one or two sites has been eliminated. The standard Gibbs energies for intrinsic binding, delta G1, delta G2, and delta G3, and cooperative interactions, delta G12 and delta G23, were determined at each condition (range 50-200 mM KCl). It is found that the dimer affinity for each of the three sites increases as [KCl] decreases, a striking result given that the monomer-dimer equilibrium shifts toward monomer formation under identical solution conditions [Koblan, K. S., & Ackers, G. K. (1991) Biochemistry (preceding paper in this issue)]. The magnitudes of ion-linked effects are found to differ at the three operator sites, while the intrinsic interaction binding free energies for sites OR1 and OR3 change in parallel over the entire range of [KCl]. The KCl dependencies at OR1 and OR3 represent the average release of 3.7 +/- 0.6 and 3.8 +/- 0.6 apparent ions, respectively. By contrast, the KCl dependency of OR2 binding corresponds to the displacement of 5.2 +/- 0.7 apparent ions. The ability of cI repressor to discriminate between the three operator sites thus appears linked to ion binding/release reactions.
采用定量DNase I足迹滴定法研究了单价盐活性对cI阻遏蛋白与其三个操纵基因位点OR的位点特异性和协同相互作用的影响。获得了阻遏蛋白二聚体与野生型OR的每个位点以及与一个或两个位点的结合已被消除的突变操纵基因模板的各个位点的结合等温线。在每种条件下（50 - 200 mM KCl范围）测定了内在结合的标准吉布斯自由能ΔG1、ΔG2和ΔG3以及协同相互作用的ΔG12和ΔG23。结果发现，随着[KCl]的降低，二聚体对三个位点中每个位点的亲和力增加，鉴于在相同溶液条件下单体 - 二聚体平衡向单体形成方向移动，这是一个惊人的结果[Koblan, K. S., & Ackers, G. K. (1991) Biochemistry（本期前一篇论文）]。发现离子连锁效应的大小在三个操纵基因位点有所不同，而位点OR1和OR3的内在相互作用结合自由能在[KCl]的整个范围内平行变化。OR1和OR3处的KCl依赖性分别代表平均释放3.7±0.6和3.8±0.6个表观离子。相比之下，OR2结合的KCl依赖性对应于5.2±0.7个表观离子的置换。因此，cI阻遏蛋白区分三个操纵基因位点的能力似乎与离子结合/释放反应有关。
