Interaction of the alpha-subunit of Escherichia coli RNA polymerase with DNA - Rigid body nature of the protein-DNA contact

The alpha -subunit of Escherichia coli RNA polymerase plays an important ro le in the activity of many promoters by providing a direct protein-DNA cont act with a specific sequence (UP element) located upstream of the core prom oter sequence. To obtain insight into the nature of thermodynamic forces in volved in the formation of this protein-DNA contact, the binding of the alp ha -subunit of E. coli RNA polymerase to a fluorochrome-labeled DNA fragmen t containing the rrnB P1 promoter UP element sequence was quantitatively st udied using fluorescence polarization. The alpha dimer and DNA formed a 1:1 complex in solution. Complex formation at 25 degreesC was enthalpy-driven, the binding was accompanied by a net release of 1-2 ions, and no significa nt specific ion effects were observed. The van't Hoff plot of temperature d ependence of binding was linear suggesting that the heat capacity change (D eltac(p)) was close to zero. Protein footprinting with hydroxyradicals show ed that the protein did not change its conformation upon protein-DNA contac t formation. No conformational changes in the DNA molecule were detected by CD spectroscopy upon protein-DNA complex formation. The thermodynamic char acteristics of the binding together with the lack of significant conformati onal changes in the protein and in the DNA suggested that the alpha -subuni t formed a rigid body-like contact with the DNA in which a tight complement ary recognition interface between alpha -subunit and DNA was not formed.