Binding affinity of Escherichia coli RNA polymerase·σ~(54) holoenzyme for the glnAp2, nifH and nifL promoters

摘要

Escherichia coli RNA polymerase associated with the σ~(54) factor (RNAP·σ~(54)) is a holoenzyme form that transcribes a special class of promoters not recognized by the standard RNA polymerase·σ~(70) complex. Promoters for RNAP·σ~(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·σ~(54) to the three promoters glnAp2, nifH and nifL. Binding studies were conducted by monitoring the changes in fluorescence anisotropy upon titrating RNAP·σ~(54) to carboxy-rhodamine-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 -Δlog(K_d)/Δlog(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.