Linkage between operator binding and dimer to octamer self-assembly of bacteriophage lambda cI repressor.
Linkage between operator binding and dimer to octamer self-assembly of bacteriophage lambda cI repressor.
复制标题
噬菌体 lambda cI 阻遏物的操纵子结合与二聚体到八聚体自组装之间的联系。
DOI：
10.1021/bi971368k
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发表时间：
1997
期刊：
影响因子：
2.9
通讯作者：
Senear,DF
中科院分区：
文献类型：
--
作者：
Rusinova,E;Ross,JB;Laue,TM;Sowers,LC;Senear,DF
噬菌体λCi抑制物二聚体与噬菌体操作者或OL的特定位点的协同结合控制着噬菌体的发育状态。长期以来，协作性一直被认为是通过抑制物二聚体的自组装形成四聚体来调节的，四聚体可以同时与相邻的操纵子结合。最近，我们证明了当自由抑制物二聚体在溶液中自结合时，四聚体是协同组装反应中的中间体，导致八聚体成为主要的高阶物种[Senear，D.F.，et al.(1993年)生物化学32,6179−6189]。即使作为组装反应中的少数组分，四聚体也可以解释成对的协同作用。以类似的方式，如果它能够同时结合所有三个操纵子，八聚体就可以解释三向协同作用。事实上，单纯基于抑制子的自组装，天真的预测是，抑制子−或相互作用应该比它们更具协作性。显然，抑制子自组装以外的过程对协作性也有不利的贡献。在这里，我们将重点放在抑制子自关联和操纵子结合之间的耦合，作为对协作性可能的不利贡献。用沉降平衡分析比较了抑制物二聚体−或1复合体和自由抑制物二聚体的二聚体−八聚体缔合反应。荧光各向异性被用来研究OR1与自由二聚体和组装成高阶物种的二聚体的结合。这些实验的结果表明，这种耦合产生了一个重要的、依赖于盐的不利贡献。由于这些实验中使用的寡核苷酸的大小与单个操纵子位点的大小相同，这种偶联是由蛋白质而不是DNA介导的。这种机制没有解释额外的、不依赖于盐的、不利的贡献，我们认为这种贡献是通过DNA传递的。因此，这两个大分子的结构转变产生的不利贡献仅用于缓和自缔合的影响。我们推测这是蛋白质与−相互作用的一个普遍特征。
Cooperative binding of the bacteriophage λcI repressor dimer to specific sites of the phage operators ORand OLcontrols the developmental state of the phage. Cooperativity has long been thought to be mediated by self-assembly of repressor dimers to form tetramers which can bind simultaneously to adjacent operators. More recently, we demonstrated that when free repressor dimers self-associate in solution, tetramer is an intermediate in a concerted assembly reaction leading to octamer as the predominant higher order species [Senear, D. F., et al. (1993)Biochemistry 32, 6179−6189]. Even as a minority component in the assembly reaction, tetramer can account for pairwise cooperativity. In a similar manner, were it able to bind all three operators simultaneously, octamer could account for three-way cooperativity. In fact, based solely on repressor self-assembly, the naive prediction is that the repressor−ORinteractions should be substantially more cooperative than they are. Evidently, there are unfavorable contributions to cooperativity from processes other than repressor self-assembly. Here, we focus on coupling between repressor self-association and operator binding as one possible unfavorable contribution to cooperativity. Sedimentation equilibrium analysis was used to compare the dimer−octamer association reactions of a repressor dimer−OR1 complex and free repressor dimer. Fluorescence anisotropy was used to investigate OR1 binding to free dimers and dimers assembled as higher order species. The results of these experiments indicate a significant and salt-dependent unfavorable contribution generated by such coupling. Since the oligonucleotides used in these experiments are the size of single operator sites, this coupling is mediated by the protein, not by the DNA. This mechanism does not account for an additional, salt-independent, unfavorable contribution which we presume is transmitted via the DNA. Thus, unfavorable contributions generated by structural transitions in both macromolecules serve to moderate the effect of self-association alone. We speculate that this is a general feature of cooperative protein−DNA interactions.