Transcriptional activation via DNA-looping: visualization of intermediates in the activation pathway of E. coli RNA polymerase·σ 54 holoenzyme by scanning force microscopy

Abstract

Scanning force microscopy (SFM) has been used to study transcriptional activation of Escherichia coli RNA polymerase·σ 54 (RNAP·σ 54) 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·σ 54 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·σ 54 at the glnA promoter; (3) association between DNA bound RNAP·σ 54 and NtrC D54E,S160F with the intervening DNA looped out; and (4) the activated open promoter complex of RNAP·σ 54. Measurements of the DNA bending angle of RNAP·σ 54 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·σ 54 closed complex to the open complex is accompanied by an increase of the DNA bending angle.