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2 Acta Biochimica Polonica

2 2001

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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

100%

Łoziński T. , Wierzchowski K. L.

tom 48

nr 2

495-510

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.

Mg2plus 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

86%

Lozinski T. , Wierzchowski K. L.

Acta Biochimica Polonica

2001

tom 48

nr 2

Footprinting studies of prokaryotic open transcription complexes (RP O), based on oxidation of pyrimidine residues by KMnO4 and/or OsO4 at a single oxidant dose, have suggested that the extent of DNA melt ing in the tran scrip tion bub ble re gion increases in the presence of Mg2+. In this work, quantitative KMnO4 footprinting in func tion of the ox i dant dose of RPo, us ing EscherichiacoliRNA poly mer ase (Es7 ) at a fully func tional syn thetic pro moter Pa hav ing -35 and -10 con sen sus hexamers, has been used to de ter mine in di vid ual rate con stants of ox i da tion of T res i dues in this region at 37° C in the ab sence of Mg2+ and in the pres ence of 10 mM MgCl2, and to eval u- ate there from the ef fect of Mg on the ex tent of DNA melt ing. Pop u la tion dis tri butions of end-labeled DNA frag ments cor re spond ing to ox i dized Ts were quan ti fied and an a lyzed ac cord ing to the sin gle-hit ki netic model. Pseudo-first or der re ac tiv ity rate con stants, kx, thus ob tained dem on strated that Mg2+ ions bound to RPo merely enhanced the re ac tiv ity of all 11ox i diz able thymines be tween the +3 and -11 pro moter sites by a po si tion-dependent fac tor: 3-4 for those lo cated close to the tran scrip tion start point +1 in ei ther DNA strand, and about 1.6 for those lo cated more dis tantly there from. On the ba sis of these ob ser va tions, we con clude that Mg2+ ions bound to RPo at Pa do not in flu ence the length of the melted DNA re gion and pro pose that the higher re ac tiv ity of thymines re sults mainly from lower lo cal re pul sive elec tro static bar ri ers to MnO4- dif fu sion around carboxylate bind ing sites in the cat a lytic cen ter of RPo and pro moter DNA phos phates.