Effect of upstream curvature and transcription factors H-NS and LRP on the efficiency of Escherichia coli rRNA promoters P1 and P2 - a phasing analysis.

To study the influence of DNA curvature and DNA-binding proteins, which interact with curved DNA on bacterial promoters, we constructed two sets of promoter variants in which a synthetic DNA-bending module was fused at defined distances and angular orientations with respect to the transcription start sites. The distance between the synthetic binding site centre and the transcription start site of the different constructs varied by up to 20 bp, corresponding to almost two complete helical B-DNA turns. The rRNA promoters rrnB P1 and rrnB P2 were selected as target promoters. While in its natural context P1 depends on upstream curved DNA and several transcription factors that bind to this region, promoter P2 is not preceded by curved DNA, nor is it believed to be directly regulated by transcription factors. In vitro transcription measurements of both promoters in the absence of transcription factors varied with the phase of the curved upstream DNA element, underlining the importance of DNA conformation to promoter efficiency. Specific binding of H-NS and LRP to the curved DNA element was demonstrated by gel shift and footprint analysis. Binding affinity was not notably altered for the different distance variants. We demonstrated that the two proteins acted as repressors for both promoters. The extent of H-NS-mediated repression for both promoters did not vary strongly with the phasing of the upstream binding module. In contrast, LRP-dependent repression showed a clear dependence on the angular orientation of the constructs. Phasing-dependent repression is very distinct for P2 but only rudimentary for the P1 promoter.