Chao M V, Martinson H G, Gralla J D
Biochemistry. 1980 Jul 8;19(14):3260-9. doi: 10.1021/bi00555a025.
We have shown previously that lac repressor binds specifically and quantitatively to lac operator restriction fragments which have been complexed with histones to form artificial nucleosomes (203 base pair restriction fragment) or core particles (144 base pair restriction fragment. We describe here a quantitative method for determining the equilibrium binding affinities of repressor for these lac reconstitutes. Quantitative analysis shows that the operator-histone reconstitutes may be grouped into two affinity classes: those with an affinity for repressor close to that of naked DNA and those with an affinity 2 or more orders of magnitude less than that of naked DNA. All particles in the lac nucleosome preparations bind repressor with high affinity, but the lac core particle preparations contain particles of both high and low affinities for repressor. Formaldehyde cross-linking causes all high-affinity species to suffer a 100-fold decrease in binding affinity. In contrast, there is no effect of cross-linking on species of low affinity. Therefore, the ability of a particle to be bound tightly by repressor depends on a property of the particle which is eliminated by cross-linking. Control experiments have shown that chemical damage to the operator does not accompany cross-linking. Therefore, the property sensitive to cross-linking must be the ability of the particle to change conformation. We infer that the particles of low native affinity, like cross-linked particles, are of low affinity because of an inability to facilitate repressor binding by means of this conformational change. Dimethyl suberimidate cross-linking experiments show that histone-histone cross-linking is sufficient to preclude high-affinity binding. Thus, the necessary conformational change involves a nucleosome histone core event. We find that the ability of a particle to undergo a repressor-induced facilitating conformational change appears to depend on the position of the operator along the DNA binding path of the nucleosome core. We present a general model which proposes that nucleosomes are divided into domains which function differentially to initiate conformational changes in response to physiological stimuli.
我们之前已经表明，乳糖阻遏物能特异性且定量地结合与组蛋白复合形成人工核小体（203碱基对限制片段）或核心颗粒（144碱基对限制片段）的乳糖操纵基因限制片段。我们在此描述一种定量方法，用于测定阻遏物对这些乳糖重组体的平衡结合亲和力。定量分析表明，操纵基因 - 组蛋白重组体可分为两个亲和力类别：一类对阻遏物的亲和力接近裸露DNA，另一类的亲和力比裸露DNA低2个或更多数量级。乳糖核小体制剂中的所有颗粒都以高亲和力结合阻遏物，但乳糖核心颗粒制剂中包含对阻遏物具有高亲和力和低亲和力的颗粒。甲醛交联导致所有高亲和力物种的结合亲和力下降100倍。相比之下，交联对低亲和力物种没有影响。因此，颗粒被阻遏物紧密结合的能力取决于颗粒的一种特性，而这种特性会因交联而消除。对照实验表明，交联过程中操纵基因没有化学损伤。因此，对交联敏感的特性必定是颗粒改变构象的能力。我们推断，天然亲和力低的颗粒，就像交联颗粒一样，亲和力低是因为无法通过这种构象变化促进阻遏物结合。亚胺基二甲酯交联实验表明，组蛋白 - 组蛋白交联足以排除高亲和力结合。因此，必要的构象变化涉及核小体组蛋白核心事件。我们发现，颗粒经历阻遏物诱导的促进性构象变化的能力似乎取决于操纵基因沿核小体核心DNA结合路径的位置。我们提出了一个通用模型，该模型认为核小体被分为不同功能的结构域，这些结构域在生理刺激下会引发不同的构象变化。
