Figure 2.

Exposed DNA binding surfaces suggest that σ^J adopts a conformation that can readily interact with the promoter. (A and B) Mapping of the −10 and −35 promoter DNA on σ^J₂ and σ^J₄ by superposition with previously determined structures of the −10 promoter/σ₂ (PDB ID: 4LUP) and −35 promoter/σ₄ (PDB ID: 2H27) complexes (60,63). (C) A schematic representation of DNA binding to σ^J. (D) The exposed DNA binding surfaces of σ^J₂ and σ^J₄ reveals that the Snoal_2 domain tethers the two DNA binding domains in this conformation but is unlikely to interact with the DNA. (E) Radiolabeled sigI-promoter DNA was incubated with (E) σ^J (0–6 μM) or (F) σ^JΔSnoaL_2 (0–64 μM) and separated on a non-denaturing PAGE. DNA binding is significantly reduced in σ^JΔSnoaL_2. The wedge at the top of each panel depicts the gradient of protein concentration. The concentrations of σ^J from left to right are 0, 1, 1.5, 1.75, 2, 2.25, 2.5, 2.75, 3, 3.5, 4, 5 and 6 μM respectively. Similarly, the concentration of σ^JΔSnoaL_2 from left to right are 0, 1, 2, 4, 8, 16, 32 and 64 μM respectively. The right-most lane in panel (F) is loaded with 5 μM σ^J as a binding control. (G) In surface plasmon resonance experiments varying concentrations of (G) σ^J and (H) σ^JΔSnoaL_2 were passed on a chip with an immobilized sigI-promoter. Put together, these experiments reveal that deletion of SnoaL_2 significantly impairs σ^J–promoter DNA interaction.