Shimamoto N, Utiyama H
Biochemistry. 1983 Dec 6;22(25):5869-78. doi: 10.1021/bi00294a028.
The highly cooperative binding of fd gene 5 to single-stranded DNA was studied kinetically by rapid photo-cross-linking and stopped-flow UV absorption measurements. The observed change in absorbance was shown to be due to the binding by direct evidence of rapid photo-cross-linking of the bound proteins to fd DNA. The bimolecular rate constant obtained for the association was 1.6 X 10(10) M-1 s-1 (in terms of the molecular concentration of DNA), which was concluded to be diffusion controlled. The breakdown of cluster complexes on fd DNA was induced by the addition of large excess amounts of short single-stranded DNA. The breakdown took place in about 1 s. The kinetic process of redistribution of dissociated proteins was monitored by rapid photo-cross-linking and subsequent electrophoresis of the cross-linked complex. The dissociated proteins first formed isolated complexes, but later they were again converted into the cluster. The kinetic results showed that the cooperativity originated from the stabilization of the protein-DNA complex by the cluster formation, not from the accelerated association in the cluster formation. This kind of cooperative binding was shown to perform negative feedback control in the cluster formation. On the basis of the kinetic results obtained, we proposed a model for the regulatory role of the fd gene 5 protein in the synthesis of single-stranded fd DNA.
通过快速光交联和停流紫外吸收测量，对fd基因5与单链DNA的高度协同结合进行了动力学研究。通过结合蛋白与fd DNA快速光交联的直接证据表明，观察到的吸光度变化是由于结合所致。缔合的双分子速率常数为1.6×10¹⁰ M⁻¹ s⁻¹（以DNA的分子浓度计），据推断这是受扩散控制的。加入大量过量的短单链DNA可诱导fd DNA上簇状复合物的分解。分解在大约1秒内发生。通过快速光交联和交联复合物的后续电泳监测解离蛋白重新分布的动力学过程。解离的蛋白首先形成孤立的复合物，但随后又再次转化为簇状。动力学结果表明，协同性源于簇状形成对蛋白质-DNA复合物的稳定作用，而非源于簇状形成过程中的加速缔合。这种协同结合在簇状形成中表现出负反馈控制。基于所获得的动力学结果，我们提出了一个关于fd基因5蛋白在单链fd DNA合成中的调控作用的模型。
