Scanning force microscopy (SFM) has been used to study transcriptional activation of Escherichia coli RNA polymerase·σ54 (RNAP·σ54) at the glnA promoter by the constitutive mutant NtrC(D54E,S160F) of the NtrC Protein (nitrogen regulatory protein C). DNA-protein complexes were deposited on mica and images were recorded in air. The DNA template was a 726 bp linear fragment with two NtrC binding sites located at the end and about 460 bp away from the RNAP·σ54 glnA promoter. By choosing appropriate conditions the structure of various intermediates in the transcription process could be visualized and analyzed: (1) different multimeric complexes of NtrC(D54E,S160F) dimers bound to the DNA template; (2) the closed complex of RNAP·σ54 at the glnA promoter; (3) association between DNA bound RNAP·σ54 and NtrC(D54E,S160F) with the intervening DNA looped out; and (4) the activated open promoter complex of RNAP·σ54. Measurements of the DNA bending angle of RNAP·σ54 closed promoter complexes yielded an apparent bending angle of 49(±24)°. Under conditions that allowed the formation of the open promoter complex, the distribution of bending angles displayed two peaks at 50 (± 24)°and 114 (± 18)°, suggesting that the transition from the RNAP·σ54 closed complex to the open complex is accompanied by an increase of the DNA bending angle.