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Multiple Site‐Specific Binding of Fis Protein to Escherichia coli nuoA‐N Promoter DNA and its Impact on DNA Topology Visualised by Means of Scanning Force Microscopy

The control of bacterial DNA topology is one of the general means to coordinating gene activity as a function of environmental changes. Among the few “histone-like” proteins, which are involved in architectural changes of prokaryotic DNA, Fis (factor for inversion stimulation), a small protein (98 aa/12 kD), acts on DNA in a growth phase-dependent manner. The regulatory response of Fis is associated with a substantial increase in protein concentration during the exponential growth phase of E. coli. The Fis protein is involved in different biological processes in E. coli that enhance site-specific recombination, control DNA replication and regulate transcription of a number of genes. For instance, the protonor Na-pumping NADH dehydrogenase I of E. coli is encoded by the nuoA-N operon, which comprises a regulatory region with two promoters, P1 and P2, located upstream of the coding sequence. Under respiratory growth conditions, nuo expression is stimulated in the early exponential growth phase and to a smaller extent in the stationary growth phase. The stimulation in the early exponential phase is affected mainly by the Fis protein. Motivated by these findings, the effect of relevant transcriptional regulator (Fis) on nuoA-N expression has been investigated by using the respective regulatory mutants of previous work. By DNAseI footprinting assays, three Fis binding sites were identified. The observation that Fis can change the overall shape of supercoiled DNA molecules provides an important clue to the possible role of Fis in controlling the architecture of prokaryotic chromosomal DNA in exponentially growing bacterial cells. To achieve a deeper understanding of the structural organisation and the interactions between the nuo promoter and the site-specific binding protein, Fis, we focused on the direct visualisation of Fis–DNA complexes by scanning force microscopy (SFM). Scanning force microscopy is particularly well suited since, as well as unambiguous identification of the location of the proteins by direct visualisation of the protein–DNA complex, the determination of the oligomeric state of the protein is possible by estimating the volume of the protein aggregate and the topology of the DNA. SFM has already been proven to be a powerful tool for the structural analysis of different protein–DNA complexes at high resolution without the need of external contrast enhancement. Hence, SFM analysis of DNA–Fis complexes would provide direct evidence that the protein has a strong impact on the general conformation of DNA. Our objective was twofold. On the one hand, we wanted to investigate whether binding is specific and if up to three Fis dimers bind to the proposed three binding sites in the region of the nuo promoter. On the other hand, our goal was to investigate the impact of Fis on the architecture of DNA. Analysis of DNA scission of Fis–DNA by a collection of Fis conjugates to 1,10-phenanthroline copper combined with comparative gel electrophoresis provides evidence that a bending angle between 508 and 908 occurs due to flanked DNA wrapping around the bound Fis molecules. It is possible to determine the degree of compaction due to wrapping of the DNA around the protein complexes. For this purpose, we compared the contour length of the DNA in the absence and presence of Fis. Figure 1 displays the 1242 bp dsDNA fragment that was used in this study and contains the