Protein-induced DNA bending clarifies the architectural organization of the σ54-dependent glnAp2 promoter

sigma(54)-RNA polymerase (E sigma(54)) predominantly contacts one face of the DNA helix in the closed promoter complex, and interacts with the upstream enhancer-bound activator via DNA looping. Up to date, the precise face of E sigma(54) that contacts the activator to convert the closed complex to an open one remains unclear. By introducing protein-induced DNA bends at precise locations between upstream enhancer sequences and the core promoter of the sigma(54)-dependent glnAp2 promoter without changing the distance in-between, we observed a strong enhanced or decreased promoter activity, especially on linear DNA templates in vitro. The relative positioning and orientations of E sigma(54), DNA bending protein and enhancer-bound activator on linear DNA were determined by in vitro footprinting analysis. Intriguingly, the locations from which the DNA bending protein exerted its optimal stimulatory effects were all found on the opposite face of the DNA helix compared with the DNA bound E sigma(54) in the closed complex. Therefore, these results provide evidence that the activator must approach the E sigma(54) closed complexes from the unbound face of the promoter DNA helix to catalyse open complex formation. This proposal is further supported by the modelling of activator-promoter DNA-E sigma(54) complex.