Beckmann P, Martin S R, Lane A N
Laboratory of Molecular Structure, National Institute for Medical Research, The Ridgeway, Mill Hill, London.
Eur Biophys J. 1993;21(6):417-24. doi: 10.1007/BF00185869.
The interaction of the trp repressor with several trp operator DNA fragments has been examined by DNA gel retardation assays and by circular dichroism, in the absence and presence of the corepressor L-tryptophan. The holorepressor binds stoichiometrically to both the trpO and aroH operators, forming 1:1 complexes. In the presence of excess protein, additional complexes are formed with these operator fragments. The relative electrophoretic mobilities of the 1:1 complexes differ significantly for trp and aroH operators, indicating that they differ substantially in gross structure. A mutant trp operator, trpOc, has low affinity for the holorepressor, and forms only complexes with stoichiometries of 2:1 (repressor: DNA) or higher, which have a very low electrophoretic mobility. Specific binding is also accompanied by a large increase in the intensity of the near ultraviolet circular dichroism, with only a small blue shift, which is consistent with significant changes in the conformation of the DNA. Large changes in the chemical shifts of three resonances in the 31P NMR spectrum of both the trp operator and the aroH operator occur on adding repressor only in the presence of L-tryptophan, consistent with localised changes in the backbone conformation of the DNA.
在有无辅阻遏物L-色氨酸存在的情况下，通过DNA凝胶阻滞分析和圆二色性研究了色氨酸阻遏物与几个色氨酸操纵基因DNA片段的相互作用。全阻遏物以化学计量比与色氨酸操纵基因（trpO）和芳香族氨基酸羟化酶操纵基因（aroH）结合，形成1:1复合物。在蛋白质过量的情况下，这些操纵基因片段会形成额外的复合物。trpO和aroH操纵基因的1:1复合物的相对电泳迁移率有显著差异，表明它们在总体结构上有很大不同。一种突变的色氨酸操纵基因trpOc对全阻遏物的亲和力较低，只形成化学计量比为2:1（阻遏物:DNA）或更高的复合物，其电泳迁移率非常低。特异性结合还伴随着近紫外圆二色性强度的大幅增加，只有很小的蓝移，这与DNA构象的显著变化一致。仅在L-色氨酸存在的情况下加入阻遏物时，trp操纵基因和aroH操纵基因的31P NMR谱中三个共振峰的化学位移发生了很大变化，这与DNA主链构象的局部变化一致。
