DNA bending caused by the binding of AraC to DNA
In this work we studied the structure of the AraC/DNA complex using AFM to measure the DNA bend angle caused by the binding of AraC to thearaI1 -araI2 region of DNA. AFM is able to produce a direct view of bending at the single molecule level without the need for labeling the DNA or protein. Independently of the measurements, we built a model using known AraC domain structures, biochemical contacts between AraC and DNA, and modifications of the conformation of DNA in order to be consistent with all dimensions. The measured DNA bend angle and the one predicted in the model agree within the uncertainties. We believe that the model shows the likely overall positions of the basic components (DDs, DBDs, DNA) of AraC bound to DNA.
The measured bend angle distribution for AraC/DNA complexes has a peak at 69° ± 25°. A possible reason for the broad distribution is that the 7-residue linker of the DBD bound to I2 is flexible, leading to a range of angles. Experimental evidence indicates that the interdomain linker is highly flexible and most likely not an alpha helix.6 In vivo , AraC binds tightly to the two direct repeat half-site sequences araI1-araI2. Experiments also show that AraC can bind tightly to inverted (araI1 -invaraI1 ) DNA half-sites, indicating the ability of the linker to change its coiling conformation. Another possible reason for the broad distribution is that the AraC/DNA complexes would land on the mica substrate in different ways; a collapse of DNA onto mica could appear in the AFM scan to have a range of bend angles.
All of our bend angle distributions have a peak at 0°. There could be unbound DNA on the mica surface. Using a dissociation constant Kd = 2 x 10-12 M for AraC – DNA binding in the presence of arabinose,27 calculations show that prior to deposition on mica, all of the DNA should be bound to AraC. However in the final deposition and washing steps, it is possible that some of the DNAs became unbound. In addition, AraC/DNA complexes might have landed on the surface in an orientation where the DNA would appear straight (Figure 7C). If that were the case, one would expect that the structure would have a higher height where the DNA and protein overlap. We did not observe this effect and cannot make a definitive statement due to the presence of aggregates and the noise level in the images.
We can speculate on the role of DNA bending. AraC is a transcription regulatory protein, and like other DNA binding proteins, AraC causes DNA to bend. Perez-Martin and Espinosa28 proposed that increases in DNA curvature would facilitate RNA polymerase-promoter interactions during the initiation of transcription. In the arabinose operon in E. coli , upon the addition of arabinose, AraC binds toaraI1 and araI2 , and AraC and cyclic AMP receptor protein (CRP) both help RNA polymerase (RNAP) bind to the pBADpromoter as well as speed the formation of open complex to initiate transcription.29, 30 We conjecture that substantial bending of the DNA binding sites of CRP, AraC, and RNAP would allow a compact structure to form that could involve specific contacts among the three proteins and improve protein-DNA interactions.15Bending of DNA may also assist melting of the double-stranded structure in the region where transcription is to begin, like sharp bending of a rope separates its strands.
A schematic diagram is shown in Figure 8 of CRP, AraC, RNAP and DNA. The dashed lines show a potential path of DNA where CRP has induced a DNA bend angle of 80°, AraC a bend of 69°, and RNAP a bend in between 30°-90°, as suggested by experimental data.31, 32, 33As can be seen, somewhat greater bending anywhere in the complex could bring CRP and RNAP into contact. The bending of DNA by AraC appears to facilitate the process of forming a compact structure.