Liu Y C, Matthews K S
Department of Biochemistry and Cell Biology, Rice University, Houston, Texas 77251-1892.
J Biol Chem. 1994 Jan 21;269(3):1692-8.
We have examined the interaction of a series of mutant trp repressors with various operator DNA sequences using gel retardation. Binding to 40 base pairs (bp) TrpEDCBA operator yielded patterns distinct from the wild-type protein for superrepressors EK13, EK18, and EK49, with a protein-DNA complex of higher stoichiometry (three dimers/operator) than observed for wild-type repressor (two dimers/operator). This higher stoichiometry complex may contribute to the enhanced binding affinity and higher protein-operator stability observed for the superrepressors. In contrast, DN46 displayed the same complexes characteristic of the wild-type protein, although the complex of a single dimer with operator was more prominent in the DN46 binding pattern than wild-type despite higher apparent affinity of this protein for TrpEDCBA operator than wild-type protein. The binding of AV77 was indistinguishable from the wild-type protein. Similar patterns to that found for TrpEDCBA were also observed for the 40-bp aroH operator and symmetrized derivatives of TrpEDCBA for these superrepressors. Binding of EK13, EK18, and EK49 superrepressors to half-site DNAs, composed of 20 bp of TrpEDCBA sequence coupled with 20 bp of lac operator sequence, yielded 2:1 complex as the primary product with no detectable 3:1 complex; thus, two half-sites appear to be required for generation of the 3:1 complex. Mutation in the tryptophan-binding site can also generate higher order complexes with TrpEDCBA DNA as demonstrated by the binding of VA58; the presence of 3:1 complex with this protein was also dependent on the presence of two half-sites. In addition to effects of sequence changes in the protein, the ligand employed can influence the binding pattern, as demonstrated for EK49 and VA58 using 5-methyl-tryptophan; the 3:1 complex is produced more prominently and at lower protein concentration for both mutants. It is apparent from these data that binding of the trp repressor to DNA is influenced by the operator sequence, the nature of the corepressor, as well as interactions (perhaps involving the N-terminal regions) that occur within and between the dimeric structure of this protein.
我们使用凝胶阻滞分析法研究了一系列突变型色氨酸阻遏物与各种操纵子DNA序列之间的相互作用。与40个碱基对（bp）的TrpEDCBA操纵子结合时，超阻遏物EK13、EK18和EK49产生的模式与野生型蛋白不同，其蛋白质-DNA复合物的化学计量比（三个二聚体/操纵子）高于野生型阻遏物（两个二聚体/操纵子）。这种更高化学计量比的复合物可能有助于超阻遏物增强的结合亲和力和更高的蛋白质-操纵子稳定性。相比之下，DN46显示出与野生型蛋白相同的复合物特征，尽管与操纵子结合的单个二聚体复合物在DN46的结合模式中比野生型更突出，尽管该蛋白对TrpEDCBA操纵子的表观亲和力高于野生型蛋白。AV77的结合与野生型蛋白无法区分。对于40-bp的aroH操纵子以及这些超阻遏物的TrpEDCBA对称衍生物，也观察到了与TrpEDCBA类似的模式。EK13、EK18和EK49超阻遏物与由20 bp的TrpEDCBA序列和20 bp的lac操纵子序列组成的半位点DNA结合时，产生的主要产物是2:1复合物，未检测到3:1复合物；因此，似乎需要两个半位点才能产生3:1复合物。色氨酸结合位点的突变也可以与TrpEDCBA DNA形成高阶复合物，如VA58的结合所示；该蛋白3:1复合物的存在也依赖于两个半位点的存在。除了蛋白质序列变化的影响外，所使用的配体也可以影响结合模式，如使用5-甲基色氨酸对EK49和VA58的研究所示；对于这两个突变体，3:1复合物在较低的蛋白质浓度下更显著地产生。从这些数据可以明显看出，色氨酸阻遏物与DNA的结合受到操纵子序列、辅阻遏物的性质以及该蛋白二聚体结构内部和之间发生的相互作用（可能涉及N端区域）的影响。
