3.1 AFM results
DNA consisting of I1-spacer-I2 was deposited on a mica substrate and imaged with AFM. For 302 bp DNA, a comparison is shown for a DNA-only sample (Figure 3A) and samples prepared with DNA, AraC, and arabinose (Figures 3B, C). For DNA-only, most structures have a curvilinear shape. When AraC was added, DNA exhibited a sharper bend in many structures, where, in most of the cases, the bend was occurring about one-third of the length from one end, consistent with the asymmetric placement of I1-spacer-I2 within the 302 bp sequence. For simple structures, the interior angle φ of the bend was measured by drawing tangents to the contour on both sides of the bend, as shown in Figure 4A. Because the 302 bp DNA was so short, the tangents were effectively constructed near the ends of the DNA. The DNA bend angle θ was calculated from θ = 180° - φ. For each θ histogram, a Gaussian function was fit to all nonzero θ. The average bend angle and standard deviation for 302 bp DNA-only samples was 47° ± 27° (Figure 5B). When AraC and arabinose were added, θ = 75° ± 26° (Figure 5C); DNA bending was stronger.
DNA bend angles were also measured for 560 bp DNA. In comparison with 302 bp DNA (Figure 3A), 560 bp DNA contained more wiggles (Figure 3D), and it was not possible to determine exactly where to measure a bend angle. However, when AraC and arabinose were added to 560 bp DNA, many structures showed a pronounced checkmark shape (Figures 3E, F). A histogram was constructed for simple structures resulting in θ = 65° ± 21° (Figure 5D). This average for 560 bp AraC/DNA complexes was somewhat less than for 302 bp AraC/DNA, suggesting that the longer length may assist with reducing the bend angle when DNA is deposited on a mica substrate. Because the θ values for 302 bp and 560 bp overlapped significantly, we combined the bend angle data for both (Figure 5A) and obtained θ = 69° ± 25°.
Many types of DNA structures appeared on the mica substrates. The circular features in Figs. 3B, C, E, F represent AraC, AraC/DNA complexes, or aggregates. For AraC/DNA samples, the heights of the circular features were .12-.47 nm, suggesting that the shorter features were protein while the taller ones were aggregates. For DNA, criteria had to be established on what to measure. We decided to not measure U- and J-shaped structures (Figure 4B) as well as segmented structures (Figure 4C). In many cases, there was a small hook at the end of the DNA (Figure 4A) or a small circular feature (Figure 4C); these were ignored. Shorter DNA fragments also appeared perhaps due to impurities or incomplete synthesis in the PCR reactions. For 302 bp and 560 bp DNA, we did not measure structures shorter in length than 80 nm and 135 nm, respectively.
The question arises whether the protein is visible in the AraC/DNA structure. Due to AFM tip broadening effects and noise in the images, we cannot conclusively state whether the protein is visible. Shorter circular features on the mica, which may be protein, are in the same height range as most of the DNA. Based on the model (see below), we would expect that the protein would not lie on top of the DNA; instead it would be near the corner of a checkmark next to the DNA.
For 560 bp DNA, the average length for DNA samples was 159 ± 24 nm (Figure S2), shorter than what would be expected in the presence of NiCl2 and MgCl2 (see Discussion). Uncertainties in the length measurements could arise from AFM tip broadening and imprecise contour determination.