Binding affinity of Escherichia coli RNA polymerase•σ54 holoenzyme for the glnAp2, nifH and nifL promoters

Escherichia coli RNA polymerase associated with the sigma(54) factor (RNAP.sigma(54)) is a holoenzyme form that transcribes a special class of promoters not recognized by the standard RNA polymerase.sigma(70) com plex. Promoters for RNAP.sigma(54) vary in their overall 'strength' and show differences in their response to the presence of DNA curvature between enhancer and promoter. In order to examine whether these effects are related to the promoter affinity, we have determined the equilibrium dissociation constant K-d for the binding of RNAP.sigma(54) to the three promoters glnAp2, nifH and nifL. Binding studies were conducted by monitoring the changes in fluorescence anisotropy upon titrating RNAP.sigma(54) to carboxyrhodamine-labeled DNA duplexes. For the glnAp2 and nifH promoters similar values of K-d = 0.94 +/- 0.55 nM and K-d = 0.85 +/- 0.30 nM were determined at physiological ionic strength, while the nifL promoter displayed a significantly weaker affinity with K-d = 8.5 +/- 1.9 nM. The logarithmic dependence of K-d on the ionic strength I was -Deltalog(K-d)/Deltalog(I) = 6.1 +/- 0.5 for the glnAp2, 5.2 +/- 1.2 for the nifH and 2.1 +/- 0.1 for the nifL promoter. This suggests that the polymerase can form fewer ion pairs with the nifL promoter, which would account for its weaker binding affinity.