Imaging of IHF-induced DNA Bending Structure by AFM

Abstract

Integration host factor (IHF) of Escherichia coli is a DNA-bending protein. Although first discovered as a host factor for bacteriophage λ integration, IHF functions in many processes that involve higher order protein-DNA complexes: e.g., in replication, where it binds to ori C; in transcriptional regulation, where it binds upstream of many σ-dependent promoters; and in a variety of site-specific recombination systems. The primary function of IHF appears to be architectural, i.e., introducing a sharp bend in the DNA that facilitates the interaction of other components in a nucleoprotein array. In this study, the structure of the DNA-IHF nucleoprotein complex in the OP1 promoter region on the Pseudomonas TOL plasmid was analyzed using atomic force microscopy (AFM), which can directly visualize biological structure under near-native conditions without crystallization. We found that IHF induced a DNA bend in the promoter regulatory region of OP1 upon its binding, supporting the DNA-loop model for the activation of OP1 transcription. The DNA fragment containing the OP1 promoter was purified from the palsmid, and it was amplified by PCR. The IHF protein was purified from E. coli strain D1210HP transformed with the plasmid pPL-hip him A-5. The IHF-DNA complexes were formed by mixing the both of solutions, and proteins not reacted with DNA fragments were removed by filtration. The NanoScopeIIIa Multi Mode system operated in tapping mode was used for observation of IHF-DNA complexes. The AFM image of the IHF-DNA complexes showed a sharp DNA bend at the IHF-binding site on the fragment. This sharp DNA bend can be used as a clear landmark of IHF-binding site on the templates. To investigate whether the binding of IHF to the IHF binding site induced bending of the template, a statistical analysis on images of IHF-DNA complexes was performed. Measurements of the DNA bending angles yielded a distribution with the mean bend angle of 123 °. Contrary, uncomplexed DNA molecules showed Gaussian distribution centered at 0 °, indicating no intrinsic curvature of the DNA fragments at these locations. This suggests that the bending observed on the complexed DNA molecules was dependent on the binding of IHF. The observed bend-angle distribution in the absence of IHF is to be accounted for by random thermal fluctuations. As shown in this study, AFM will be a useful tool for visualizing the effects of protein-DNA interactions and contribute to a better understanding of the complex mechanisms of transcriptional regulation.