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Mg 2+ ‐modulated KMnO 4 reactivity of thymines in the open transcription complex reflects variation in the negative electrostatic potential along the separated DNA strands
Author(s) -
Łoziński Tomasz,
Wierzchowski Kazimierz L.
Publication year - 2005
Publication title -
the febs journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 204
eISSN - 1742-4658
pISSN - 1742-464X
DOI - 10.1111/j.1742-4658.2005.04705.x
Subject(s) - thymine , dna , chemistry , nucleotide , kinetics , stereochemistry , rna polymerase , ion , escherichia coli , transcription (linguistics) , thymidine , crystallography , biochemistry , organic chemistry , gene , physics , linguistics , philosophy , quantum mechanics
There is still a controversy over the mechanism of promoter DNA strand separation upon open transcription complex (RPo) formation by Escherichia coli RNA polymerase: is it a single or a stepwise process controlled by Mg 2+ ions and temperature? To resolve this question, the kinetics of pseudo‐first‐order oxidation of thymine residues by KMnO 4 in the −11 … +2 DNA region of RPo at the λP R promoter was examined under single‐hit conditions as a function of temperature (13–37 °C) in the absence or presence of 10 m m MgCl 2 . The reaction was also studied with respect to thymidine and its nucleotides (TMP, TTP and TpT) as a function of temperature and [MgCl 2 ]. The kinetic parameters, ox k and ox E a , and Mg‐induced enhancement of ox k proved to be of the same order of magnitude for RPo–λP R and the nucleotides. Unlike the complex, ox E a for the nucleotides was found to be Mg‐independent. The isothermal increase in ox k with increasing [Mg 2+ ] was thus interpreted in terms of a simple model of screening of the negative charges on phosphate groups by Mg 2+ ions, lowering the electrostatic barrier to the diffusion of MnO 4 – anions to the reactive double bond of thymine. Similar screening isotherms were determined for the oxidation of two groups of thymines in RPo at a consensus‐like Pa promoter, differing in the magnitude of the Mg effect. Together, the findings show that: (a) the two DNA strands in the −11…+2 region of RPo–λP R are completely separated over the whole range of temperatures investigated (13–37 °C) in the absence of Mg 2+ (b) Mg 2+ ions induce an increase in the rate of the oxidation reaction by screening negatively charged phosphate and carboxylate groups; and (c) the observed thymine reactivity and the magnitude of the Mg effect reflect variation in the strength of the electrostatic potential along the separated DNA strands, in agreement with the current structural model of RPo.