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), bas ed on oxidation of pyrimidine residues by KMnO4 and/or OsO4 at a single oxi dant dose, have suggested that the extent of DNA melting in the transcripti on bubble region increases in the presence of Mg2+ In this work, quantitati ve KMnO4 footprinting in function of the oxidant dose of RPO, using Escheri chia coli RNA polymerase (E sigma (70)) at a fully functional synthetic pro moter 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 d egreesC in the absence of Mg2+ and in the presence of 10 mM MgCl2, and to e valuate on the extent of DNA melting. Population distributions of end-label ed DNA fragments corresponding to oxidized Ts were quantified and analyzed according to the single-hit kinetic model. Pseudo-first order reactivity ra te constants, k(i), 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 locat ed close to the transcription start point +1 in either DNA strand, and abou t 1.6 for, those located more distantly therefrom. On the basis of these ob servations, 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 barrier s to MnO4- diffusion around carboxylate binding sites in the catalytic cent er of RPO and promoter DNA phosphate.