Determination of the DNA-binding kinetics of three related but heteroimmune bacteriophage repressors using EMSA and SPR analysis
Determination of the DNA-binding kinetics of three related but heteroimmune bacteriophage repressors using EMSA and SPR analysis
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DOI：
10.1093/nar/gkm172
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发表时间：
2007-05-01
影响因子：
14.9
通讯作者：
Haggard-Ljungquist, Elisabeth
中科院分区：
文献类型：
--
作者：
Henriksson-Peltola, Petri;Sehlen, Wilhelmina;Haggard-Ljungquist, Elisabeth
噬菌体P2、P2、Hy dis和W phi非常相似，但异种免疫的大肠杆菌噬菌体。结构基因有96%以上的同源性，但抑制因子的同源性在43%到63%之间。此外，包含两个直接重复序列的操纵子在序列、长度、相对于启动子的位置和直接重复之间的间距上都是不同的。我们用凝胶迁移率改变分析(EMSA)比较了野生型和突变操纵子在体内对基因表达的影响，并用表面等离子共振(SPR)分析比较了抑制子与野生型或突变操纵子形成的复合体对蛋白质-DNA相互作用的实时动力学。利用EMSA，阻遏子形成了不同的蛋白质-DNA复合体，只有W-Phi受到点突变的显著影响。然而，SPR分析显示，P2和W Phi算符突变体的缔合速率常数降低，解离速率常数增大。P2-Hy-dis的缔合速率常数过快，无法测定。P2的Hy分解反应曲线为三相，而P2和W Phi C均为两相。因此，在这些密切相关的噬菌体之间，形成复合体的动力学和形成的复合体的性质有很大的不同。
Bacteriophages P2, P2 Hy dis and W Phi are very similar but heteroimmune Escherichia coli phages. The structural genes show over 96% identity, but the repressors show between 43 and 63% identities. Furthermore, the operators, which contain two directly repeated sequences, vary in sequence, length, location relative to the promoter and spacing between the direct repeats. We have compared the in vivo effects of the wild type and mutated operators on gene expression with the complexes formed between the repressors and their wild type or mutated operators using electrophoretic mobility shift assay (EMSA), and real-time kinetics of the protein-DNA interactions using surface plasmon resonance (SPR) analysis. Using EMSA, the repressors formed different protein-DNA complexes, and only W Phi was significantly affected by point mutations. However, SPR analysis showed a reduced association rate constant and an increased dissociation rate constant for P2 and W Phi operator mutants. The association rate constants of P2 Hy dis was too fast to be determined. The P2 Hy dis dissociation response curves were shown to be triphasic, while both P2 and W Phi C were biphasic. Thus, the kinetics of complex formation and the nature of the complexes formed differ extensively between these very closely related phages.