tailieunhanh - Báo cáo khoa học: Mg2+-modulated KMnO4 reactivity of thymines in the open transcription complex reflects variation in the negative electrostatic potential along the separated DNA strands

There is still a controversy over the mechanism of promoter DNA strand separation upon open transcription complex (RPo) formation by Escheri-chia 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 KMnO4 in the )11 +2 DNA region of RPo at thekPRpromoter was examined under single-hit conditions as a function of temperature (13–37 C) in the absence or presence of 10 mmMgCl2 | iFEBS Journal Mg2 -modulated KMnO4 reactivity of thymines in the open transcription complex reflects variation in the negative electrostatic potential along the separated DNA strands Footprinting of Escherichia coli RNA polymerase complex at the kPR promoter revisited Tomasz Lozinski and Kazimierz L. Wierzchowski Department of Biophysics Institute of Biochemistry and Biophysics Polish Academy of Sciences Warszawa Poland Keywords Escherichia coli RNA polymerase open transcription complex permanganate footprinting thymine oxidation kPR promoter. Correspondence K. L. Wierzchowski Department of Biophysics Institute of Biochemistry and Biophysics Polish Academy of Sciences Pawinskiego 5a 02-106 Warszawa Poland Fax 48 22 658 3646 Tel 48 22 658 4729 E-mail klw@ Website http Received 24 January 2005 revised 29 March 2005 accepted 6 April 2005 doi 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 Mg2 ions and temperature To resolve this question the kinetics of pseudo-first-order oxidation of thymine residues by KMnO4 in the -11 . 2 DNA region of RPo at the kPR promoter was examined under single-hit conditions as a function of temperature 13-37 C in the absence or presence of 10 mM MgCl2. The reaction was also studied with respect to thymidine and its nucleotides TMP TTP and TpT as a function of temperature and MgCl2 . The kinetic parameters oxk and oxEa and Mg-induced enhancement of oxk proved to be of the same order of magnitude for RPo .PR and the nucleotides. Unlike the complex oxEa for the nucleotides was found to be Mg-independent. The isothermal increase in oxk with increasing Mg2 was thus interpreted in terms of a simple model of screening of the negative charges on phosphate groups by Mg2 ions lowering the electrostatic barrier to the diffusion of

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