Takeda Y, Kim J G, Caday C G, Steers E, Ohlendorf D H, Anderson W F, Matthews B W
J Biol Chem. 1986 Jul 5;261(19):8608-16.
The mode of interaction of Cro repressor with specific and nonspecific sites on DNA was explored by chemical modification and protection of lysine and tyrosine residues. Cro has 8 lysines. In the presence of DNA, lysines 32 and 56 are fully protected and lysines 21, 62, and 63 are partially protected from alkylation. However, the terminal amino group and lysines 8, 18, and 39 are not protected. Location of the protected and unprotected lysines on the three-dimensional Cro structure defines a DNA-binding region. The results provide direct experimental support for a mode of interaction between Cro and DNA, in which Cro buries its 2-fold related alpha-helices in consecutive DNA major grooves (Anderson, W. F., Ohlendorf, D. H., Takeda, Y., and Matthews, B. W. (1981) Nature 290, 754-758; Ohlendorf, D. H., Anderson, W. F., Fisher, R. G., Takeda, Y., and Matthews, B. W. (1982) Nature 298, 718-723). In the model, the carboxyl-terminal part of Cro was tentatively presumed to interact with the DNA minor groove. Protection of lysines 62 and 63 confirms the involvement of the carboxyl terminus in DNA binding. Although nonspecific and specific DNA protect the same lysine residues, there are differences in the nature of the interaction of Cro with nonspecific and specific DNA. Cro-nonspecific DNA interaction is salt-sensitive, suggesting that the interaction is predominantly electrostatic. On the other hand, Cro-specific DNA interaction is salt-resistant, suggesting that the interaction may include nonelectrostatic components (hydrogen bonds and hydrophobic interactions) as well. Protection experiments of tyrosine residues (against iodination) suggest that the conformation of Cro repressor changes in two stages: first, when Cro binds at nonspecific sites, and, second, when Cro binds to specific sites on DNA.
通过对赖氨酸和酪氨酸残基的化学修饰及保护，研究了Cro阻遏蛋白与DNA上特异性和非特异性位点的相互作用模式。Cro有8个赖氨酸。在DNA存在的情况下，赖氨酸32和56受到完全保护，赖氨酸21、62和63受到部分保护而免受烷基化作用。然而，末端氨基以及赖氨酸8、18和39未受到保护。在Cro的三维结构上受保护和未受保护的赖氨酸的位置确定了一个DNA结合区域。这些结果为Cro与DNA之间的相互作用模式提供了直接的实验支持，即Cro将其2倍相关的α螺旋埋入连续的DNA大沟中（安德森，W.F.，奥伦多夫，D.H.，武田，Y.，和马修斯，B.W.（1981年）《自然》290，754 - 758；奥伦多夫，D.H.，安德森，W.F.，费舍尔，R.G.，武田，Y.，和马修斯，B.W.（1982年）《自然》298，718 - 723）。在该模型中，暂时假定Cro的羧基末端部分与DNA小沟相互作用。赖氨酸62和63的保护证实了羧基末端参与DNA结合。虽然非特异性和特异性DNA保护相同的赖氨酸残基，但Cro与非特异性和特异性DNA相互作用的性质存在差异。Cro - 非特异性DNA相互作用对盐敏感，表明这种相互作用主要是静电作用。另一方面，Cro - 特异性DNA相互作用对盐具有抗性，表明这种相互作用可能还包括非静电成分（氢键和疏水相互作用）。酪氨酸残基的保护实验（针对碘化）表明，Cro阻遏蛋白的构象在两个阶段发生变化：第一，当Cro结合在非特异性位点时；第二，当Cro结合到DNA上的特异性位点时。
