Nucleic Acids Research Advance Access published online on April 4, 2007
Nucleic Acids Research, doi:10.1093/nar/gkm172
Molecular Biology |
Determination of the DNA-binding kinetics of three related but heteroimmune bacteriophage repressors using EMSA and SPR analysis
Department of Genetics, Microbiology and Toxicology, Stockholm University, S-106 91 Stockholm, Sweden
*To whom correspondence should be addressed. Tel: +46 8 161270; Fax: +46 8 164315; Email: Elisabeth.Haggard{at}gmt.su.se
Received August 11, 2006. Revised March 5, 2007. Accepted March 6, 2007.
Bacteriophages P2, P2 Hy dis and W
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 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 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 C were biphasic. Thus, the kinetics of complex formation and the nature of the complexes formed differ extensively between these very closely related phages.