Structure and Dynamics of the IL2RA Gene Promoter Figure 3. AFM imaging of DNA loop, GTF, nucleosome and HMGA1 positioning on the IL2RA gene promoter. (A) AFM imaging in liquid of DNA loop positioning along the 1290 bp IL2RA fragment (upper right panel) and the 898 bp IL2RA fragment (lower right panel) and statistical analysis (left panel) of their positioning given by the loop middle point (green vertical bars) from N = 21 AFM images. (B) AFM imaging of TBP-TFIIB positioning along the 563 bp IL2RA fragment (right panels) and statistical analysis (left panel) of their positioning (orange vertical bars) from N = 51 AFM images. (C) AFM imaging of mononucleosome positioning along the 898 bp IL2RA fragment (right panels) and statistical analysis (left panel) of dyad positioning (blue vertical bars) from N = 100 AFM images. (D) AFM imaging of HMGA1 positioning along the 898 bp IL2RA fragment (right panels) and statistical analysis (left panel) of dyad positioning (blue vertical bars) from N = 73 molecules. doi:10.1371/journal.pone.0018811.g003 nucleosomes formation was shown to be disfavored (Figure 3C) leaving DNA accessible to other protein binding factors. Note however some slight overlap with the inhibitory nucleosome positioned on the TATA box and TSS (see Discussion). Discussion What controls the TSS until transcription initiation? The melting of DNA at the correct places is essential to recruit RNA Pol II and to initiate gene transcription. This opening is known to be directed by the helicase activity of TFIIH proteins [47] but the intrinsic structural and mechanical properties of DNA also play an important role. Thus, although a very low Gibbs free energy DG value in the TSS vicinity would be advantageous because less energy would be needed to open the double helix, it would increase the risk of uncontrolled melting. Actually, opening location is likely to result from multiple non-exclusive causes. In the IL2RA promoter region, the region of less thermal stability (very low DG) extends over the PRRII control element (Figure 2G) where the architectural HMGA1 TFs and Elf-1 are expected to bind and to regulate inducible transcription of the IL2RA gene [39]. The increased stability observed inward the TSS is indicative of the specificity of transcription initiation since more energy would be required to open DNA. Interestingly, similar DG profiles with the TSS located in a ramp between an upstream low DG region and a rather high DG region at the 59-end of the gene, are shared by the ribosomal gene promoters [5] and by the yem-a TATA-less RNA Pol II promoter [48]. The DNA structure and in particular the supercoiled conformation theoretically predicted (Figure 1) and experimentally observed (Figure 2) in the PRRII region could also help preventing DNA strand separation at the TSS. It could be argued that the supercoil found on the IL2RA molecules (Figure 2, Figures S6 and S7) was not intrinsic to the DNA sequence but induced by the interaction with the substrate. PLoS ONE | www.plosone.org 6 April 2011 | Volume 6 | Issue 4 | e18811