CRP fixes the rotational orientation of covalently closed DNA molecules

Abstract

Five minicircles of 284 bp were constructed with a reporter sequence located approximately opposite a CRP binding site. The spacing between the center of the CRP site and this sequence is varied within 1.2 helical turns. The reactivity of the reference sequence to DNAse I was determined on the minicircles and on the corresponding fragments, in both the absence and presence of CRP. A rigorous mathematical analysis of the data shows that in the absence of CRP no preferred rotational orientation of the DNA is observed. In contrast, binding of CRP fixes, in a phase-dependent manner, the rotational orientation of the reporter sequence in the minicircles. This result illustrates the transmission at a distance along the DNA molecule of a structural modification. Such effects modulate the extent of synergy between activators and polymerases during the initiation of transcription.