Henriksson-Peltola Petri, Sehlén Wilhelmina, Haggård-Ljungquist Elisabeth
Department of Genetics, Microbiology and Toxicology, Stockholm University, S-106 91 Stockholm, Sweden.
Nucleic Acids Res. 2007;35(10):3181-91. doi: 10.1093/nar/gkm172. Epub 2007 Apr 4.
Bacteriophages P2, P2 Hy dis and WPhi 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 WPhi was significantly affected by point mutations. However, SPR analysis showed a reduced association rate constant and an increased dissociation rate constant for P2 and WPhi 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 WPhi C were biphasic. Thus, the kinetics of complex formation and the nature of the complexes formed differ extensively between these very closely related phages.
噬菌体P2、P2 Hy dis和WPhi是非常相似但具有异源性免疫的大肠杆菌噬菌体。其结构基因显示出超过96%的同一性，但阻遏蛋白的同一性在43%至63%之间。此外，包含两个直接重复序列的操纵基因在序列、长度、相对于启动子的位置以及直接重复序列之间的间距方面存在差异。我们使用电泳迁移率变动分析（EMSA）比较了野生型和突变型操纵基因对基因表达的体内影响，以及阻遏蛋白与野生型或突变型操纵基因形成的复合物，并使用表面等离子体共振（SPR）分析了蛋白质 - DNA相互作用的实时动力学。使用EMSA时，阻遏蛋白形成了不同的蛋白质 - DNA复合物，只有WPhi受到点突变的显著影响。然而，SPR分析显示P2和WPhi操纵基因突变体的缔合速率常数降低，解离速率常数增加。P2 Hy dis的缔合速率常数太快而无法确定。P2 Hy dis的解离反应曲线显示为三相，而P2和WPhi C均为双相。因此，在这些密切相关的噬菌体之间，复合物形成的动力学以及所形成复合物的性质存在广泛差异。
