Senear D F, Batey R
Department of Molecular Biology and Biochemistry, University of California, Irvine 92717.
Biochemistry. 1991 Jul 9;30(27):6677-88. doi: 10.1021/bi00241a007.
The effects of proton and KCl activity on the nonspecific lambda cI repressor-DNA interactions and on the site-specific repressor-O(R) interactions were compared, in order to assess their roles in site specificity. The repressor-O(R) interactions were studied by using DNase I footprint titration. The Gibbs free energy changes for binding and for cooperativity were determined between 25 and 300 mM KCl, from individual-site isotherms for the binding of repressor to O(R) and to reduced-valency mutants. The proton-linked effects on repressor-O(R) interactions have been published [Senear, D. F., & Ackers, G. K. (1990) Biochemistry 29, 6568-6577; Senear, D. F., & Bolen, D. W. (1991) Methods Enzymol. (in press)]. Nonspecific binding was studied by using a nitrocellulose filter binding assay, which proved advantageous in this case, due to the relatively weak nonspecific binding, and precipitation of repressor-DNA complexes. Filter binding provided measurements at low binding density where precipitation did not occur. The data provide estimates of the Gibbs free energy changes for nonspecific, intrinsic binding, but not for cooperativity. The KCl concentration dependencies of the intrinsic binding constants indicate that ion release plays similar roles in distinguishing between the operators and in discriminating operator from nonoperator DNA. Binding to DNA is accompanied by net proton absorption. Near neutral pH, proton linkages to operator and nonoperator binding are the same. Differences at acid and at basic pH implicate the same ionizable repressor groups in distinguishing between the operators and in discriminating operator from nonoperator DNA. The results indicate similar overall modes of operator and nonoperator binding of repressor, but implicate indirect effects of DNA sequence as important contributors to sequence recognition.
比较了质子和氯化钾活性对非特异性λ cI阻遏蛋白与DNA相互作用以及对位点特异性阻遏蛋白与O(R)相互作用的影响，以评估它们在位点特异性中的作用。通过使用DNase I足迹滴定法研究阻遏蛋白与O(R)的相互作用。根据阻遏蛋白与O(R)以及与低价突变体结合的单一位点等温线，测定了25至300 mM氯化钾浓度下结合和协同作用的吉布斯自由能变化。关于质子对阻遏蛋白与O(R)相互作用的影响已有报道[塞内尔，D.F.，& 阿克斯，G.K.（1990年）《生物化学》29卷，6568 - 6577页；塞内尔，D.F.，& 博伦，D.W.（1991年）《酶学方法》（即将出版）]。通过使用硝酸纤维素滤膜结合测定法研究非特异性结合，在这种情况下这被证明是有利的，因为非特异性结合相对较弱，且阻遏蛋白 - DNA复合物会沉淀。滤膜结合在低结合密度下进行测量，此时不会发生沉淀。这些数据提供了非特异性、内在结合的吉布斯自由能变化的估计值，但未提供协同作用的估计值。内在结合常数对氯化钾浓度的依赖性表明，离子释放在位点区分以及区分位点与非位点DNA方面起着相似的作用。与DNA的结合伴随着净质子吸收。在接近中性pH值时，质子与位点和非位点结合的联系是相同的。在酸性和碱性pH值下的差异表明，相同的可电离阻遏基团在位点区分以及区分位点与非位点DNA方面起作用。结果表明阻遏蛋白在位点和非位点结合的总体模式相似，但暗示DNA序列的间接效应是序列识别的重要因素。
