DIFFUSION-DRIVEN MECHANISMS OF PROTEIN TRANSLOCATION ON NUCLEIC-ACIDS .2. THE ESCHERICHIA-COLI REPRESSOR-OPERATOR INTERACTION - EQUILIBRIUM MEASUREMENTS
DIFFUSION-DRIVEN MECHANISMS OF PROTEIN TRANSLOCATION ON NUCLEIC-ACIDS .2. THE ESCHERICHIA-COLI REPRESSOR-OPERATOR INTERACTION - EQUILIBRIUM MEASUREMENTS
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DOI：
10.1021/bi00527a029
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发表时间：
1981-01-01
期刊：
影响因子：
2.9
通讯作者：
VONHIPPEL, PH
中科院分区：
文献类型：
--
作者：
WINTER, RB;VONHIPPEL, PH
研究了大肠杆菌 lac 阻遏物与操纵子位点的平衡结合，作为一价盐浓度、包含操纵子的 DNA 分子长度和（通过使用各种天然 lac 假操纵子）操纵子碱基对序列的函数。硝化纤维滤膜测定用于直接获得阻遏蛋白-操纵因子 [RO] 缔合常数 (KRO) 值以及缔合与解离速率常数的比率 (ka/Kd)。 KRO 的测量是在没有 Mg2+ 或其他二价离子的情况下进行的，从而可以直接估计静电（电荷-电荷）相互作用对 RO 复合物稳定性的贡献。对含有已知大小的 lac 操纵子的 DNA 限制性片段进行了一项研究。 RO 相互作用的大小取决于盐浓度。 log KRO 与 log [KCl] 的关系图在 0.1-0.2 M KCl 范围内呈线性，从该图的斜率可以确定 RO 复合物的形成涉及 6-7 个电荷-电荷相互作用。该值与操作符类型和片段 > .apprx 的 DNA 片段大小无关。长度为 170 个碱基对。这个电荷-电荷相互作用的数量明显少于 RD [阻遏蛋白-非操纵基因 DNA] 复合物形成中涉及的 11 个此类相互作用，表明阻遏蛋白以不同的构象与操纵基因和非操纵基因 DNA 结合。 RO 相互作用涉及结合自由能的大量 (> 50%) 非静电成分，与 RD 相互作用相反，RD 相互作用的所有结合自由能本质上都是静电的。当在不同的 DNA 片段上测量时，辅助（lacZ 基因）伪算子的结合常数 (KRO2) 比主要（生理）算子的 KRO1 弱 5 倍。当两个操作员都在同一条 DNA 上时，KRO2 的测量值为 .apprx。比KRO1小25倍。 KRO3，第三级（I 基因）伪算子的结合常数，在 KRO1 .simeq 的盐浓度下估计为 < 1010 M-1。 1013 M-1。在相同的环境条件下，与短DNA片段结合的阻遏蛋白KRO1比与长DNA片段结合的KRO1小。其中一些发现以及文献中的其他发现表明对 RO 结合常数的长期影响；讨论了这种效应的可能分子基础。这些测量为 RO 动力学结合机制的分析提供了平衡基础，并且还允许比较操纵子、伪操纵子和非操纵子 DNA 的阻遏物结合亲和力。这些结果进一步证明了周围（非特异性）DNA 在控制 RO 复合物的平衡稳定性以及形成和解离速率方面的重要性。
The equilibrium binding of E. coli lac repressor to operator sites was studied as a function of monovalent salt concentration, of length of the DNA molecule containing the operator, and (by using various natural lac pseudooperators) of operator base pair sequence. The nitrocellulose filter assay was used to obtain values of repressor-operator [RO] association constants (KRO), both directly and as ratios of association to dissociation rate constants (ka/Kd). Measurements of KRO were made in the absence of Mg2+ or oher divalent ions, allowing a direct estimate of the contribution of electrostatic (charge-charge) interactions to the stability of the RO complexes. A study was made of lac operator-containing DNA restriction fragments of known size. The magnitude of the RO interaction is salt concentration dependent. A plot of log KRO vs. log [KCl] is linear over the 0.1-0.2 M KCl range, and from the slope of this plot, it can be determined that RO complex formation involves 6-7 charge-charge interactions. This value is independent of operator type and of DNA fragment size for fragments > .apprx. 170 base pairs in length. This number of charge-charge interactions is appreciably less than the 11 such interactions involved in RD [repressor-nonoperator DNA] complex formation, suggesting that repressor binds to operator and to nonoperator DNA in different conformations. The RO interaction involves a substantial (> 50%) nonelectrostatic component of the binding free energy, in contrast to the RD interaction for which all the binding free energy appears to be electrostatic in nature. The binding constant (KRO2) for the secondary (lacZ gene) pseudooperator is 5-fold weaker than KRO1 for e primary (physiological) operator when both are measured on separate pieces of DNA. When both operators are on the same piece of DNA, the measured value of KRO2 is .apprx. 25-fold smaller than that of KRO1. KRO3, the binding constant for the tertiary (I gene) pseudooperator, was estimated to be < 1010 M-1 at salt concentrations where KRO1 .simeq. 1013 M-1. KRO1 for repressor binding to short DNA fragments is smaller than that for binding to long DNA fragments under the same environmental conditions. Several of these findings, together with others in the literature, are suggestive of long-range effects on RO binding constants; possible molecular bases for such effects are discussed. These measurements provide the equilibrium underpinnings of this analysis of RO kinetic binding mechanisms and also allow comparisons of repressor binding affinities for operator, pseudooperator and nonoperator DNA. These results further demonstrate the importance of the surrounding (nonspecific) DNA in controlling the equilibrium stability and the rates of formation and dissociation of RO complexes.