Tài liệu Báo cáo khoa học: Mg2+-modulated KMnO4 reactivity of thymines in the open transcription

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 Łozin´ ski and Kazimierz L. Wierzchowski

Department of Biophysics, Institute of Biochemistry and Biophysics, Polish Academy of Sciences, Warszawa, Poland

Transcription initiation in prokaryotes involves specific

recognition between the )10 and )35 conserved hexa￾mers of the promoter DNA and RNA polymerase

holoenzyme followed by large and concerted conform￾ational changes in both components of the binary

complex leading to separation of the template and

nontemplate strands from )11 to
+4 bp, relative to

the transcription start point, and formation of the

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,

Pawin´ skiego 5a, 02-106 Warszawa, Poland

Fax: +48 22 658 3646

Tel: +48 22 658 4729

E-mail: [email protected]

Website: http://www.ibb.waw.pl

(Received 24 January 2005, revised 29

March 2005, accepted 6 April 2005)

doi:10.1111/j.1742-4658.2005.04705.x

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 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 magni￾tude for RPo–kPR 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 MnO4

– anions to the

reactive double bond of thymine. Similar screening isotherms were deter￾mined 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–kPR are completely separated over the whole range of temperatures

investigated (13–37
C) in the absence of Mg2+ (b) Mg2+ 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.

Abbreviations

R or RNAP, RNA polymerase; P, promoter; RPo, open transcription complex; Thd, thymidine; TpT, dithymidine (3¢-5¢)-monophosphate.

2838 FEBS Journal 272 (2005) 2838–2853 ª 2005 FEBS