Can a transcriptional activator known to bend DNA be functionally replaced by a sequence‐directed bend in Escherichia coli? To investigate this question, a partially truncated promoter was used, deleted of its ‐35 region and of its CRP binding site, leaving only two Pribnow boxes as functional elements. Synthetic and naturally occurring curved DNA sequences introduced upstream from these elements could restore transcription at either one of the two natural starts. Some of these hybrid promoters turned out to be more efficient than the CRP activated wild‐type gal promoter in vivo. Control experiments performed with very similar sequences devoid of any curvature produced weak promoters only. Minimal changes in the location of the centre of curvature or perturbation in the amount of curvature strongly affected the level of expression. No significant stimulation of transcription could be detected in vitro. Furthermore, both gal P1 and P2 starts could be activated in vivo but also in vitro via a properly positioned CRP binding site. This partial analogy suggests that bending induced by the cAMP‐CRP complex upon binding to its site may be biologically relevant to the mechanism of transcriptional activation.

A series of gal promoter mutants has been used to compare the in vitro selectivities of the two forms of Escherichia coli RNA polymerase, Ea38 and EaY70. In the absence of the CRP-cAMP complex, E-38 shows a strong preference for the gaRI1 promoter, whereas Ea70 preferentially initiates transcription from the gaP2 promoter. Ea8 selectivity is not affected by the nature and position of the upstream sequences or by the phasing between synthetic upstream curved sequences and the-10 regions. In fact, all effects of mutations in the extended-10 region can be accounted for without evoking strong new sequence preferences for Ea38. Finally, both E-38 and Ea70 initiate transcription from the gaR1 promoter in the presence of CRP-cAMP complex and support direct cAMP-CRP activation at several CRP-dependent promoters.

Replacement of the CRP-binding site of the gal control region by curved sequences can lead to the restoration of promoter strength in vivo. One curved sequence called 5A6A, however, failed to do so. The gene hns exerts a strong negative control on the resulting 5A6A gal promoter as well as on the distant bla promoter, specifically in a 5A6A gal context. The product of this gene, H-NS, displays a better affinity for this particular insert compared to other curved sequences. Mechanisms by which H-NS may repress promoters both at short and long distances from a favoured binding site are discussed.