Johnson G G, Geiduschek E P
Biochemistry. 1977 Apr 5;16(7):1473-85. doi: 10.1021/bi00626a036.
The interaction of the phage SPO1 protein transcription factor 1 (TF1), with DNA has been analyzed by membrane filter binding and by sedimentation methods. Substantially specific binding of TF1 to helical SPO1 DNA can be demonstrated by nitrocellulose filter-binding assays at relatively low ionic strength (0.08). However, TF1-DNA complexes dissociate and reequilibrate relatively rapidly and this makes filter-binding assays unsuitable for quantitative measurements of binding equilibra. Accordingly, the sedimentation properties of TF1-DNA complexes have been explored and a short-column centrifugation assay has been elaborated for quantitative measurements. Preferential binding of TF1 to the hydroxymethyluracil-containing SPO1 DNA has also been demonstrated by short-column centrifugation. TF1 binds relatively weakly and somewhat cooperatively to SPO1 DNA at many sites; TF1-DNA complexes dissociate and reequilibrate rapidly. At 20 degrees C in 0.01 M phosphate, pH 7.5, 0.15 KC1, one molecule of TF1 can bind to approximately every 60 nucleotide pairs of SPO1 DNA.
已通过膜滤结合法和沉降法分析了噬菌体SPO1蛋白转录因子1（TF1）与DNA的相互作用。在相对较低的离子强度（0.08）下，通过硝酸纤维素滤膜结合试验可证明TF1与螺旋状SPO1 DNA存在显著的特异性结合。然而，TF1-DNA复合物解离并重新平衡的速度相对较快，这使得滤膜结合试验不适用于结合平衡的定量测量。因此，已对TF1-DNA复合物的沉降特性进行了探索，并精心设计了一种短柱离心试验用于定量测量。短柱离心法也证明了TF1对含羟甲基尿嘧啶的SPO1 DNA具有优先结合。TF1在许多位点与SPO1 DNA的结合相对较弱且有一定协同性；TF1-DNA复合物解离并重新平衡迅速。在20℃、0.01M磷酸盐、pH 7.5、0.15KCl条件下，一个TF1分子大约可与SPO1 DNA的每60个核苷酸对结合。
