Mg2+ ions do not induce expansion of the melted DNA region in the open complex formed by Escherichia coli RNA polymerase at a cognate synthetic Pa promoter. A quantitative KMnO4 footprinting study

Footprinting studies of prokaryotic open transcription complexes (RPO), based on oxidation of pyrimidine residues by KMnO4 and/or OsO4 at a single oxidant dose, have suggested that the extent of DNA melting in the transcription bubble region increases in the presence of Mg2+. In this work, quantitative KMnO4 footprinting in function of the oxidant dose of RPO, using Escherichia coli RNA polymerase (Eσ70 ) at a fully functional synthetic promoter Pa having -35 and -10 consensus hexamers, has been used to determine individual rate constants of oxidation of T residues in this region at 37°C in the absence of Mg2+ and in the presence of 10 mM MgCl2, and to evaluate therefrom the effect of Mg2+ on the extent of DNA melting. Population distributions of end-labeled DNA fragments corresponding to oxidized Ts were quantified and analyzed according to the single-hit kinetic model. Pseudo-first order reactivity rate constants, ki, thus obtained demonstrated that Mg2+ ions bound to RPO merely enhanced the reactivity of all 11 oxidizable thymines between the +3 and -11 promoter sites by a position-dependent factor: 3-4 for those located close to the transcription start point +1 in either DNA strand, and about 1.6 for those located more distantly therefrom. On the basis of these observations, we conclude that Mg2+ ions bound to RPO at Pa do not influence the length of the melted DNA region and propose that the higher reactivity of thymines results mainly from lower local repulsive electrostatic barriers to MnO4- diffusion around carboxylate binding sites in the catalytic center of RPO and promoter DNA phosphates.