Tài liệu 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 ppt

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 Łozinski 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@ibb.waw.pl Website: http://www.ibb.waw.pl (Received 24 January 2005, revised 29 March 2005, accepted 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 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–kPR and the nucleotides Unlike the complex, oxEa for the nucleotides was found to be Mg-independent The isothermal increase in ox k 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 determined for the oxidation of two groups of thymines in RPo at a consensuslike 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 Transcription initiation in prokaryotes involves specific recognition between the )10 and )35 conserved hexamers 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 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 ´ T Łozinski and K L Wierzchowski Open complex at kPR promoter open complex (RPo) capable of specific binding of NTP substrates and synthesis of nascent RNA [1] Kinetic–mechanistic studies on the initiation of transcription by Escherichia coli RNA polymerase (R) at a number of cognate promoters (P) – kPR [2–6], lac UV5 [7] and T7A1 [8] – on linear DNA templates showed that formation of RPo is a multistep process involving at least two kinetically significant intermediates, an initial complex (called I1 or RPc) and an intermediate one (I2 or RPi): k1 k2 k3 k1 k2 k3 R ỵ P ! I1 ! I2 ! RPo The I1 « I2 step is rate limiting, characterized by extensive conformational changes and a high free energy of activation, at which the strand separation is proposed to be nucleated at the )11th bp [1] In the next step, I2 « RPo, the latter process is completed by a downstream expansion of the nucleated transcription bubble A measurable population of the I2 form could be observed for the lac UV5 and kPR promoters on linear DNA templates only Negative supercoiling of the DNA template shifts the opening equilibrium (K3 ¼ k3 ⁄ k-3) towards RPo [3,7,9] For the kPR promoter on a negatively supercoiled plasmid, this equilibrium has been found shifted completely towards RPo over the temperature range 4–37 °C [10] According to the current molecular model of RPo [11,12], based on crystal structures of Thermus thermophilus [13] and Thermus aquaticus [14] RNA polymerase holoenzyme (free and complexed with forked DNA), the two separated DNA strands are held in protein channels formed by segments of the r70 and b, b¢ RNA polymerase subunits The molecular mechanism of DNA strand separation is still unknown, however Studies with mutant polymerases harboring deletions in these subunits seem to be very promising with respect to this problem They have led to selection of mutants forming partial melting intermediates [15,16], identification of RNA polymerase (RNAP) regions involved in melting, and demonstration that isolated b¢(1–314) and r2)3 fragments alone are able to melt an extended )10 promoter [17,18] Also the question of whether DNA melting by RNAP occurs as a concerted one-step or stepwise process controlled by temperature and Mg2+ ions [1] remains disputable In view of the absolute requirement for Mg2+ in the process of RNA synthesis by E coli RNAP [19], related primarily to the involvement of Mg2+ in the catalysis of internucleotide phosphodiester bond formation and binding of NTP substrates [20–23], a possible role of these ions in RPo formation has been FEBS Journal 272 (2005) 2838–2853 ª 2005 FEBS probed by kinetic [4] and footprinting experiments [10,24–26] A large difference between the observed (% 0.4) and expected (% 4; from that of % found for Na+ [2]) stoichiometry of Mg2+ ion uptake in the kinetically significant steps of RPo–kPR dissociation [4], led the authors to postulate a ‘fourth step’ hypothesis stating that in the absence of Mg2+ an intermediate open complex, RPo1, is formed which, upon specific binding of three Mg2+ ions, transforms to its transcription-competent form, RPo2 Strong enhancement by Mg2+ of susceptibility to KMnO4 oxidation of pyrimidine bases at the )12, +1 and +2 positions in kPR promoter DNA, found in subsequent footprinting experiments [10], led the authors to postulate Mg-induced expansion of the transcription bubble from its center to both ends, accompanying the RPo1 fi RPo2 transition Further studies of the Mg effect on the kPR DNA backbone scission by hydroxy radicals [24], generated in the Fenton reaction between Fe(EDTA)2– and H2O2, indicated that deoxyribose residues at positions close to the transcription start point react with OH in RPo2 but are relatively protected in RPo1, suggesting rather a downstream expansion of the transcription bubble upon binding of Mg2+ It has been proposed to result from greater steric accessibility and ⁄ or local reduction in negative charge density associated with DNA phosphates and carboxylates in the catalytic pocket of E coli RNAP holoenzyme in RPo2 Results of a similar study on the RPo–T7A1 complex [26], based on oxidation of thymines by KMnO4 and OsO4 and of DNA backbone scission by hydroxy radicals as a function of temperature, indicated that the transcription bubble consists of a Mg-independent part and a Mg-dependent one close to the catalytic site, both having individual transition temperatures indicative of a stepwise expansion of the melted DNA region The appearance of discrete melting intermediates in the complex formed by Bacillus subtilis RNAP at the flagellin promoter [27] was also claimed on the basis of temperature-dependent changes in the permanganate footprint pattern In all these studies, very high multiple-hit doses of the permanganate were used, and the pyrimidine oxidation was assumed to be temperature-independent To assess more reliably multiple-hit footprinting data, Tsodikov et al [28] developed a quantitative method of analysis, in which chemical probing performed as a function of either concentration of the oxidant or time exposure allows evaluation of reactivity rate constants for individual bases Application of this method to permanganate oxidation of RPo–kPR showed that of the three bases (T)4, T)3 and T+2) probed at 37 °C and °C, only the reactivity of the last one was temperature2839 ´ T Łozinski and K L Wierzchowski Open complex at kPR promoter dependent [28] Our attempts to apply this method to RPo at a synthetic Pa promoter failed, however, because of the occurrence of highly competitive oxidation reactions within the RNAP component [29], which was ignored in the method We have shown that under multiple-hit KMnO4 doses commonly used in the earlier footprinting experiments, RPo becomes completely inactivated, through severe damage by multiple oxidative lesions accumulating in both the RNAP and the melted DNA region, and partially dissociated Permanganate footprinting of RPo–Pa as a function of singlehit oxidant dose [30] showed that, in this complex, Mg2+ ions not induce any expansion of the melted DNA region, but merely increase the reactivity of all thymines in a position-dependent manner, in particular those located close to the active center of the complex, as observed previously at other promoters [10,24,26,27] In a parallel study of the rate of RPo–Pa complex dissociation as a function of [Mg2+], we have shown [31] that, in the 20–37 °C temperature range, four Mg2+ ions are involved in the equilibrium K3 ¼ k3 ⁄ k-3 (equivalent to seven Na+ ions found in the case of RPo–kPR [2]), as could be expected for a fully melted transcription bubble Our current studies on the dependence on [Mg2+] of the rate of RPo–kPR dissociation show that its course is biphasic, which may indicate involvement of ionic exchange coupled to reformation of salt bridges on the protein surface upon dissociation of wrapped DNA from the complex [32] In view of (a) the presented critical assessment of the experimental approaches used in previous footprinting studies, in particular the clearly unrealistic assumption that the underlying chemical reaction of thymine oxidation is temperature-independent, and (b) the possibility that the mechanism of transcription bubble formation may depend on the promoter sequence [1], it seemed necessary to reinvestigate permanganate oxidation of the most studied RPo–kPR complex as a function of the oxidant single-hit dose, temperature and Mg2+ concentration Here we report the results of these experiments They clearly show that the pattern of oxidation of thymines in the bubble region of RPo at kPR at 37 °C is generally similar to that determined previously by us for RPo at the Pa promoter on the same template under similar conditions [30], only the reactivity of thymines in the RPo– kPR complex appeared to be significantly higher than that of analogously located bases in RPo–Pa Moreover, oxidation of thymines was shown to exhibit temperature-dependence similar to that found for thymidine and its nucleotides (see below) We believe that the effect of Mg2+ on thymine oxidation in RPo–Pa [30] and in dsDNA [33] was due 2840 mainly to the screening of negative charges of DNA phosphates (and carboxylic groups in RPo) near the thymine residues The reduction of local negative charge by Mg2+ ions bound in the catalytic pocket of RPo–kPR was also considered by the group of Record [24] as a possible source of the increased backbone and base reactivity at the start site Additional arguments in support of this notion were obtained in this work from experiments on KMnO4 oxidation of thymine residues in thymidine (Thd), TMP, TTP and dithymidine (3¢-5¢)-monophosphate (TpT) as a function of temperature and [Mg2+] In connection with the recent model of RPo structure [11,12], we show finally that the observed reactivity of thymine and its modulation by Mg2+ reflects variation in the effective electrostatic potential along the separated DNA strands determined by charged DNA phosphates and protein groups at the walls of RNAP channels surrounding the DNA Results Oxidation of the RPo–kPR complex Thymine residues in the promoter bubble DNA region of RPo formed by E coli RNAP at the kPR promoter (see Fig for sequence), contained in the pDS3 plasmid, were oxidized by KMnO4 in the absence and presence of 10 mm MgCl2 at three selected temperatures (13 °C, 25 °C and 37 °C) as a function of the oxidant dose (x ¼ [KMnO4] · t, m · s) in the range 0.004–0.04 m · s, which is known to ensure single-hit oxidation of thymines in the melted DNA region and preservation of the original structure of the complex almost intact [29] At 10 mm MgCl2 a high occupancy of the catalytic site can be expected on the basis of micromolar value of the apparent equilibrium binding constant for Mg2+ to E coli RNAP, which can be estimated from the protective effect of Mg2+ on Fe2+-induced cleavage of the protein fragments forming this site [22] PAGE-resolved DNA products of the Klenow extension reaction, Fig Sequences of the kPR and Pa promoters studied Melted regions in RPo are shown in bold, )10 and )35 recognition hexamers are underlined, and an arrow marks the transcription start point FEBS Journal 272 (2005) 2838–2853 ª 2005 FEBS ´ T Łozinski and K L Wierzchowski Open complex at kPR promoter A B C D Fig Selected KMnO4 footprints of the melted DNA region of RPo at the kPR promoter (A) and (B) Autoradiograms of 6% polyacrylamide sequencing gels (at the right side the whole, and at the left side enlarged fragments corresponding to the melted DNA region) showing resolved 32P-end-labeled ssDNA products of the Klenow primer extension reaction carried out on the nontemplate (A) and template (B) DNA strands; doses of KMnO4 (in M · s) applied at 37 °C are indicated above lanes 2–11 Minus and plus signs indicate the absence and presence of 10 mM MgCl2 in footprinting reactions Lane 1, footprint of dsDNA without RNA polymerase (C) and (D) Fragments of autoradiograms of 6% polyacrylamide sequencing gels showing resolved 32P-end-labeled ssDNA products of the Klenow primer extension reaction carried out on the nontemplate (C) and template (D) DNA strands of RPo oxidized at different temperatures and KMnO4 doses (indicated above the lanes); along the leftmost lanes positions of DNA bands corresponding to thymines in the melted region of the kPR promoter are indicated Some bands ascribed to particular oxidized thymines are doubled: the stronger component corresponds to the DNA extension reaction products ending at thymine diglycol, and the weaker one, to fragments shorter by one base formed when the extension reaction encountered oxidized thymine hydrolyzed to the ureido form corresponding to oxidized thymine, are exemplified in Fig Inspection of the gels shows that DNA bands corresponding to all thymines in the )11 … +2 promoter region came up clearly in the footprints obtained even at 13 °C and the lowest oxidant dose applied and in the absence of MgCl2 In the footprints from reactions carried out in the presence of 10 mm MgCl2, all these bands are merely more intense Note that, neither for the four cytosines present in the bubble region (at positions )1, )2, )5 and )6) nor for C)12, found oxidized under multiple-hit conditions [10], can a DNA band corresponding to FEBS Journal 272 (2005) 2838–2853 ª 2005 FEBS oxidized base be traced even at the highest oxidant dose applied and 37 °C The extent of thymine oxidation in the template and nontemplate DNA strands was evaluated (as described in Experimental procedures) by quantification of the corresponding 32P-end-labeled primer-extended DNA fragments in the footprints The average fractions of oxidized thymine thus obtained, oxfi(x), rose monoexponentially as a function of the applied oxidant dose x, as expected for a pseudo-first-order reaction This is exemplified in Fig for reactions performed at 37 °C The corresponding rate constants of the reaction, oxki, 2841 ´ T Łozinski and K L Wierzchowski Open complex at kPR promoter Fig Kinetics of KMnO4 oxidation at 37 °C of thymines in the melted region of RPo at the kPR promoter Data points (mean values: n ¼ 3, calculated standard errors in the range of 10–15%) corresponding to DNA fractions of oxidized thymines (oxfi) and unoxidized DNA (fuDNA) in the template (left column), and nontemplate (right column) strands, in the absence (j) and presence (d) of 10 mM MgCl2 were obtained by quantification of the footprints (exemplified in Fig 2) as described in Experimental Procedures; solid lines represent fitted functions (Eqn 1, oxki values in Table 1) were thus obtained by nonlinear weighted fit of Eqn (1) to the experimental data: ox fi ¼ À expðÀox ki  xÞ ð1Þ They are collected in Table The pseudo-first-order character of the oxidation reaction testifies that all thymines in RPo under the conditions studied are solvent accessible and that the flux of MnO4– anions to the reaction sites within protein channels is high enough to sustain this type of kinetics for the bimolecular reaction 2842 At 37 °C and 10 mm Mg2+, that is under conditions in which a transcription competent RPo–kPR complex has been shown to be the dominant species with fully separated DNA strands [10,24], the most reactive thymines were T)11 and T)8 of the template (t) strand and T)3 and T)4 of the nontemplate (nt) strand, whereas T+1 (t) and T+2 (nt), located close to the catalytic center, were much less reactive The least reactive, however, proved to be T)7 and T)10 of the nt strand, which are known to be involved in specific interactions with region 2.3 of r70 [1] and T)9 FEBS Journal 272 (2005) 2838–2853 ª 2005 FEBS ´ T Łozinski and K L Wierzchowski Open complex at kPR promoter Table Pseudo-first-order rate constants (oxki) for thymine oxidation by KMnO4 in the melted DNA region of RPo at the kPR promoter The mean ± SEM values of oxki were determined from nonlinear weighted least squares fit of Eqn (1) to the oxfi(x) data (Fig 3) obtained as described in Experimental procedures nt, Nontemplate DNA strands; t, template DNA strand Temperature oxki (M)1Ỉs)1) oxki,Mg (M)1Ỉs)1) Thymine (°C) [Mg2+] ¼ [Mg2+] ¼ 10 mM T+2 (nt) 37 T-3 (nt) T-4 (nt) T-7 & T-10 (nt) T-11 (t) T-9 (t) T-8 (t) T+1 (t) T+2 (nt) 25 T-3 (nt) T-4 (nt) T-7 & T-10 (nt) T-11 (t) T-9 (t) T-8 (t) T+1 (t) T+2 (nt) 13 T-3 (nt) T-4 (nt) T-7 & T-10 (nt) T-11 (t) T-9 (t) T-8 (t) T+1 (t) ox ki,Mg ⁄ oxki 3.0 7.0 6.1 0.8 ± ± ± ± 0.1 0.2 0.2 0.1 10.5 15.9 10.4 1.0 ± ± ± ± 0.2 0.5 0.4 0.1 3.5 2.3 1.7 1.25 ± ± ± ± 0.2 0.1 0.1 0.1 14.8 0.9 9.5 1.2 1.5 4.0 3.4 0.5 ± ± ± ± ± ± ± ± 0.3 0.1 0.2 0.1 0.1 0.1 0.1 0.1 23.3 1.4 16.8 5.2 7.8 10.8 6.7 0.9 ± ± ± ± ± ± ± ± 0.5 0.1 0.4 0.1 0.1 0.2 0.2 0.1 1.6 1.6 1.8 4.3 5.2 2.7 2.0 1.8 ± 0.05 ± 0.2 ± 0.05 ± 0.4 ± 0.35 ± 0.1 ± 0.1 ± 0.4 10.2 0.9 7.6 0.5 0.7 2.7 2.2 0.3 ± ± ± ± ± ± ± ± 0.2 0.1 0.1 0.1 0.1 0.1 0.1 0.1 19.6 1.1 15.5 2.6 5.5 7.3 4.8 0.6 ± ± ± ± ± ± ± ± 0.3 0.1 0.3 0.1 0.1 0.1 0.1 0.1 1.9 1.2 2.0 5.2 7.9 2.7 2.2 2.0 ± ± ± ± ± ± ± ± 0.05 0.4 0.05 1.1 1.1 0.1 0.15 0.75 6.4 0.9 5.8 0.4 ± ± ± ± 0.1 0.1 0.1 0.1 13.9 0.9 11.7 2.6 ± ± ± ± 0.2 0.2 0.2 0.2 2.2 1.0 2.0 6.5 ± ± ± ± 0.05 0.25 0.05 1.7 of the t strand The patterns of thymine reactivity in the presence and absence of Mg2+ were generally similar In the absence of Mg2+, the reactivity of all the thymines was merely lower A comparison of the corresponding oxki and oxki,Mg values shows that thymine reactivity in the presence of Mg2+ becomes enhanced by a position-dependent factor, oxki,Mg ⁄ oxki, with the largest values of 4.4 for T+1 and 3.5 for T+2, and much smaller, in the range 1.6–2.3, for the most reactive groups including T)3, T)4, T)8 and T)11 For the least reactive, T)9, the Mg effect was similar to that of the last group, whereas for T)7 and T)10 its value of 1.2 was distinctly smaller The pattern of relative thymine reactivity did not change significantly on lowering the temperature from 37 °C to 25 °C and 13 °C; only the rate constants of oxidation became progressively smaller, as would be expected for a chemical reaction, and the Mg effect became somewhat larger, for T+1 and T+2 in particular The Arrhenius plots of ln(oxki,Mg) and ln(oxki) vs ⁄ T (K) were linear (Fig 4, correlation coefficient )0.98 or better) as if oxki reflected mainly the temperature dependence of the oxidation reaction The energies of activation, oxEa, calculated from these plots (Table 2) for reactions carried out in the absence of Mg2+ were 6–8 kcalỈmol)1 (25.1–33.5 kJỈmol)1) for T)3, T)4, T)7, T)10 and T)11, distinctly higher at % 11 kcalỈmol)1 (% 46 kJỈmol)1) for T+2 and T+1, and much lower at 3.7 kcalỈmol)1 (15.5 kJỈmol)1) for T)8 The higher values of oxEa for the oxidation of T+1 and T+2, which are located close to the active center of RPo, correlate with the lower reactivity of these bases For the reactions carried out in the presence of Mg2+, the corresponding values of oxEa,Mg proved to be generally smaller for all thymines The largest decrease, by a factor of % 2, was found for T+2, and T)7 and T)10 The reactivity of T)9, which apparently did not change with temperature in the absence of Mg2+, varied in the same way as T)8 in the presence of Mg2+ Fig Effect of temperature and Mg2+ on the rate constants of thymine oxidation by KMnO4 in RPo at the kPR promoter, in Thd and TMP Arrhenius plots of oxki data (Tables and 3) in the absence (j) and presence (d) of 10 mM MgCl2; solid lines represent fitted functions, corresponding activation energies of the reaction in Table FEBS Journal 272 (2005) 2838–2853 ª 2005 FEBS 2843 ´ T Łozinski and K L Wierzchowski Open complex at kPR promoter Table Energies of activation of the KMnO4-oxidation reaction (oxEa) of thymines in the melted DNA region in RPo at the kPR promoter, and in Thd and TMP The mean ± SEM values of oxEa were determined from linear weighted least-squares fit of the Arrhenius equation to the oxki data from Table and Table ox ox Substrate Ea (kcalặmol)1) [Mg2+] ẳ Ea,Mg (kcalặmol)1) [Mg2+] ẳ 10 mM T+2 (nt) T-3 (nt) T-4 (nt) T-7 & T-10 (nt) T-11 (t) T-9 (t) T-8 (t) T+1 (t) Thd TMP 10.7 ± 0.1 7.2 ± 1.0 7.7 ± 0.9 7.2 ± 0.1 6.2 ± 0.2 3.7 ± 0.1 11.1 ± 3.3 8.6 ± 0.3 7.7 ± 0.2 4.8 ± 0.1 5.7 ± 0.1 5.5 ± 0.6 3.0 ± 1.7 4.0 ± 0.7 3.5 ± 0.3 2.8 ± 0.8 7.2 ± 3.2 8.4 ± 0.3 8.1 ± 0.5 [Mg2+] ¼ 50 mM 8.3 ± 0.1 ox Ea,Mg ⁄ oxEa 0.45 0.79 0.71 0.42 0.64 – 0.78 0.65 0.98 1.05 ± ± ± ± ± 0.03 0.11 0.23 0.24 0.11 ± ± ± ± 0.22 0.35 0.11 0.07 There is no doubt that temperature also influences the structural dynamics of RPo and may thus induce some local conformational changes in the complex affecting the accessibility of reaction centers to the oxidant This may apply for instance to T+1 and T+2 located in a more structurally rigid motif of RPo The measured values oxEa should thus be regarded as apparent The values of oxEa,Mg are formally smaller because the magnitude of the Mg effect increases progressively as the temperature falls (Table 1), which may be due to increased binding of Mg2+ This point is dealt with further in the Discussion Oxidation of thymine in thymidine and its nucleotides The kinetics of oxidation of thymine by KMnO4 [34] in thymidine, TMP, TTP and TpT was studied as a function of [MgCl2] in the range 0–100 mm in the presence of 100 mm KCl (Figure and Table 3) The pseudo-first-order rate constants of the reaction in the absence of Mg2+ appeared to be of similar magnitude to those determined for thymines in RPo–kPR under similar salt and temperature conditions (Tables and 2) For the nucleotides, they were smaller than for the parent nucleoside (21.1 m)1Ỉs)1) and decreased with the increase in negative charge on the phosphate group in the order TpT (14.4 m)1Ỉs)1), TMP (8.5 m)1Ỉs)1) and TTP (6.4 m)1Ỉs)1) In agreement with the hypothesis referred to above, the rate of Thd oxidation was independent of the 2844 Fig Effect of [MgCl2] on the kinetics of thymine oxidation by KMnO4 In TpT (A), TMP (B), TTP (C), and in two nontemplate DNA strand regions: T+2, T+3 (d) and T)2, T)3, T)4 (j) of RPo at the Pa promoter (D) The rate constants oxk of the reaction in nucleotides were determined at 25 °C, and sums of the respective oxki in the RPo–Pa complex at 37 °C Solid lines represent fitted functions (Eqn 2), the values of the fitted parameters in Table presence of Mg2+, whereas those of TMP, TpT and TTP exhibited a dependence on [MgCl2] mimicking a binding isotherm (Fig 5), which was particularly steep for TTP It is known that Thd in aqueous solution adopts the anti conformation about the N(1)–C(1¢) glycosidic FEBS Journal 272 (2005) 2838–2853 ª 2005 FEBS ´ T Łozinski and K L Wierzchowski Open complex at kPR promoter Table Pseudo-first-order rate constants (oxk) for thymine oxidation by KMnO4 in Thd and its nucleotides The mean ± SEM values of oxk were determined from nonlinear weighted least-squares fit of a single exponential decay function to the kinetic data obtained as described in Experimental Procedures Thd TpT TMP TTP ox k (M)1ặs)1) [Mg2+] ẳ kMg (M)1ặs)1) [Mg2+] ẳ 10 mM 25 13 25 37 25 13 25 Compound Temperature (°C) 21.1 12.0 6.0 14.4 13.5 8.5 5.3 2.6 6.4 20.7 11.8 6.0 15.4 15.3 10.4 6.0 2.9 11.2 ± ± ± ± ± ± ± ± ± 0.9 0.4 0.2 0.2 0.5 0.4 0.5 0.1 0.2 bond [35] in which the C-5¢-OH hydroxy group makes close contact with the C(6)-H group of the thymine ring [36] In thymidine 5¢-phosphates, the negatively charged terminal monophosphate and triphosphate groups are thus expected to be located close to the C(5)¼C(6) double bond susceptible to MnO4– attack In solutions close to neutrality, these groups chelate only one Mg2+ ion [35] The same is expected for the diester phosphate group in TpT The ox k([Mg2+]) data for all these compounds were thus fitted to a simple model assuming a single Mg2+binding site involved in the screening of negative phosphate charges: ox kMg (M)1ặs)1) [Mg2+] ẳ 50 mM ox ± ± ± ± ± ± ± ± ± ox 0.9 0.5 0.3 0.3 0.7 0.3 0.5 0.1 0.4 20.7 12.3 6.8 3.5 ± ± ± ± 0.3 0.2 0.1 0.1 Table Fitted parameters of Mg2+-screening isotherms for KMnO4 oxidation of thymidine nucleotides and the two groups of thymine residues in RPo–Pa The parameter’s values (mean ± SEM) were determined from nonlinear weighted least-squares fit of Eqn (2) to the data points shown in Fig Compound Temperature Kscr (°C) (M)1) TpT 25 TMP TTP RPo–Pa 37 ST)4, T)3, T)2 ST+2, T+3 ox k([Mg] ¼ 0) oxk([Mg] fi 1) (M)1Ỉs)1) (M)1Ỉs)1) 25.7 ± 6.6 14.4 ± 0.1 18.3 ± 0.5 32.4 ± 5.7 8.2 ± 0.1 15.5 ± 0.5 350 ± 78 6.5 ± 0.3 13.5 ± 1.1 161 ± 64 162 ± 12 9.3 ± 1.1 21.1 ± 2.7 3.0 ± 0.1 10.9 ± 0.4 kMg ẳ oxk ỵ DfẵMg2ỵ Kscr =1 ỵ ẵMg2ỵ Š  Kscr Þg ð2Þ where Kscr is a screening constant, expected to be proportional to the corresponding thermodynamic binding constant, Kass, for Mg2+ and D ¼ oxk([Mg] fi 1) – ox k([Mg] ¼ 0) is an increment by which the initial value of oxk would increase at the saturating Mg2+ concentration Values of Kscr thus obtained at 25 °C (25.7, 32.4 and 350 m)1) for TpT, TMP and TTP, respectively (Table 4) are presumably smaller than the respective binding constants Kass, as they reflect only replacement by Mg2+ of K+ ions from solvation shells of phosphate groups leading to more effective screening of their negative charges The ratio of Kscr values for TMP and TTP of % 10 is sixfold smaller than that of the intrinsic equilibrium binding constants for UMP and UTP [37], measured at 100 mm NaCl, which is probably due to the multitude of conformations adopted by the Mg-chelating triphosphate group [35], some of which apparently not contribute significantly to the electrostatic barrier to MnO4– being considered The corresponding values of oxk([Mg] fi 1) ¼ D + ox k([Mg] ¼ 0), that is the rate constant at saturating FEBS Journal 272 (2005) 2838–2853 ª 2005 FEBS Mg2+ concentration (18.3, 15.5 and 13.5 m)1Ỉs)1) tend to approach that of 21.1 m)1Ỉs)1 measured for thymidine It is thus evident that the role played by Mg2+ in the enhancement of the reactivity of thymine towards MnO4– in thymidine phosphates is mainly electrostatic in nature Therefore, the differences in ox k([Mg] ¼ 0) between thymidine and its nucleotides reflect mostly the differences in the electrostatic barrier to diffusion of MnO4– to the reactive double bond of the thymine moiety The remaining differences between the corresponding oxk([Mg] fi 1) values can be ascribed to steric factors determined by the different sizes of the substituents In the case of TpT, intramolecular stacking of thymine residues brings the two C(5)¼C(6) bonds in close proximity, thereby increasing the probability of their attack by MnO4– and decreasing the effect of the negative phosphate charges on MnO4– diffusion by dielectric shielding The temperature dependence of the kinetics of oxidation of Thd and TMP was also investigated, and the Arrhenius energies of activation determined, in the absence of Mg2+ and the presence of selected Mg2+ 2845 ´ T Łozinski and K L Wierzchowski Open complex at kPR promoter Fig Effect of [MgCl2] on KMnO4 footprint of the melted DNA region in RPo at the Pa promoter Autoradiogram of polyacrylamide sequencing gel (6%) showing resolved 32P-end-labeled ssDNA products of the Klenow primer extension reaction carried out on the nontemplate DNA strand: at the right side whole gel, and at the left side fragment corresponding to the melted DNA region; [MgCl2] in mM is indicated above the lanes; along the leftmost lane positions of DNA bands corresponding to thymines in the melted region are indicated concentrations (Table and Fig 4) They are of the same order of magnitude as those determined for thymine oxidation in RPo–kPR The activation energies of Thd and TMP oxidation in the absence and presence of 10 mm MgCl2, and for the latter also at 50 mm MgCl2, were found to be similar within the limits of experimental error This is an important observation, confirming that the mechanism underlying the Mg2+ effect on the kinetics of nucleotide oxidation does not affect the intrinsic rate of the reaction and is due solely to the screening of the negative charge on the phosphate group, thereby diminishing the electrostatic barrier to diffusion of MnO4– to the reactive double bond of the thymine moiety Mg2+ effect on the kinetics of oxidation of the RPo–Pa complex The two separated DNA strands in RPo are held in protein channels formed by segments of the r70 and b RNAP subunits [11,12] Therefore, the thymines can be expected to experience different molecular environments depending on their location It was thus of interest to determine screening isotherms for variously positioned thymine residues, analogous to those obtained for the nucleotides in the preceding section For this experiment we used RPo at the consensus-like Pa promoter [30], which at 37 °C exhibits a very similar pattern of thymine oxidation in the ssDNA region to that observed here for the RNAP–kPR complex (Fig 7), and, unlike RPo–kPR, resists relatively high [MgCl2] in titration experiments [31] RPo formed by E coli RNAP in a buffer solution containing 100 mm KCl and varying [MgCl2] in the range 0–40 mm was oxidized at 37 °C with a KMnO4 dose of 0.01 or 0.02 m · s, found to be sufficiently low to secure singlehit conditions of oxidation within the whole range of MgCl2 concentrations applied [29] The corresponding footprints (Fig 6) were quantified, and the kinetic 2846 parameters derived as described for the RPo–kPR complex Sums of the rates of oxidation, Soxki, of the two groups of thymines in the nontemplate promoter strand –(a) T+3 and T+2 and (b) T)2, T)3 and T)4 found to differ greatly in the magnitude of the Mg2+ effect at 10 mm concentration [30] – are plotted as a function of [MgCl2] in Fig The plots resemble closely the screening isotherms obtained for the nucleotides Fitting of Eqn (2) to these data yielded a value of Kscr % 160 m)1, similar for both groups of thymines (Table 4) This confirms that the Mg2+ ions involved in the screening of negative charges associate with binding sites of similar affinity in the two bubble regions, that is most probably DNA phosphates In agreement with earlier findings [30], the two groups are characterized by very different values of the oxk([Mg] fi 1) ⁄ oxk enhancement factor of 3.7 and 2.3 for (a) and (b), respectively Consequently, the maximum value of Soxki([Mg] fi 1) for the less reactive group (a) in the absence of Mg2+ tends to approach that of the more reactive one (b), as observed for TTP and TMP The still smaller maximum reactivity of T+2 and T+3 close to the catalytic center of RPo can be thus attributed by similar token to a larger steric barrier to diffusion of permanganate anions to thymines in this region Because, even at the lowest oxidant doses applied, the RPo was found to be completely inactivated transcriptionally [29], conformational changes in the catalytic center, probably caused by oxidation of Cys454 close to the NADFDGD motif [29], may in part be responsible for the observed lower steric accessibility of the two thymines to the oxidant in both the presence and absence of Mg2+ This conclusion also applies to RPo at kPR and other promoters Discussion The results show that (a) all nine thymine residues of the template and nontemplate strands in the )11 … +2 region of RPo at a plasmid-borne kPR FEBS Journal 272 (2005) 2838–2853 ª 2005 FEBS ´ T Łozinski and K L Wierzchowski promoter are susceptible to permanganate oxidation in the temperature range 13–37 °C and (b) the corresponding reaction rate constants are of the same order of magnitude as, and exhibit similar Mg2+ and temperature dependence to, those for thymine oxidation in free thymidine nucleotides The simplest interpretation of these findings is that the DNA strands in this region are completely separated, as expected for the fully ‘open’ RPo, and the observed temperature dependence of the oxidation rate constants can be interpreted as being due, for the most part, to the inherent activation energy of the reaction Moreover, the enhancement of thymine reactivity, observed at 10 mm Mg2+ in both RPo–kPR and RPo–Pa, was shown in RPo–Pa to be a continuous function of Mg2+concentration Together, the results of single-hit permanganate footprinting of RPo–kPR not support the earlier interpretation of the Mg2+ effect in single-dose multi-hit experiments for this complex [10], which stated that Mg2+ induces in a partially opened subpopulation of RPo, called RPo1, some conformational transition leading to an extension of the melted DNA region from the center outwards and to the formation of the fully open transcription competent RPo2 complex Also stoppedflow spectrofluorimetric investigations of the kinetics of RPo formation at a synthetic promoter bearing consensus )10 and )35 and UP elements have indicated that DNA opening is not affected by Mg2+ ions [38] All these observations allow us to conclude that, in plasmid-contained Pa and kPR promoters, no stable melting intermediates can be detected by perturbing the reaction with temperature and Mg2+ This point of view finds support in the recent single-molecule DNA manipulation experiments [9] on promoter unwinding by RNAP, which showed that, in this process, there are no intermediates with lifetimes longer than % s The normal temperature-dependence of the thymine oxidation reaction demonstrated in RPo calls into question the results of earlier footprinting investigations using permanganate and ⁄ or other chemical probes, in which temperature was used to visualize the allegedly stepwise opening of stable transcription complexes under the assumption that the underlying chemical reaction is temperature-independent [10,24–28] Investigations of thymine oxidation in Thd and its nucleotides in solution have unequivocally demonstrated that the mechanism underlying the Mg effect on the reaction in nucleotides is electrostatic and consists of screening the negative phosphate charges by Mg2+, thereby reducing the electrostatic barrier to diffusion of MnO4– anions towards the reactive double bond of thymine Thus the differences in the rate constants of FEBS Journal 272 (2005) 2838–2853 ª 2005 FEBS Open complex at kPR promoter thymine oxidation in the absence of Mg2+ between nucleotides bearing variously charged and sized phosphate groups reflect the differences in the extent of the electrostatic barrier to MnO4– diffusion, whereas those extrapolated to the saturating Mg2+ concentration point to the differences in the extent of the steric barriers to this process The general similarity of the kinetic characteristics of the oxidation reaction in the nucleotide and RPo–kPR and RPo–Pa systems allows analogous interpretation of the observed differences in the rate constants for individual thymines in RPo in terms of the electrostatic and steric barriers to MnO4– diffusion Consequently, the observed differences between the rate constants of thymines variously located in the separated template and nontemplate promoter strands can be attributed to the position-dependent extent of the electrostatic and steric barriers to the diffusion of the oxidant to the reactive C(5)¼C(6) thymine double bond From this perspective, it was worth comparing the reactivity of thymines in the RPo–kPR and RPo–Pa complexes determined under the same experimental conditions This comparison is shown in Fig as column plots of oxk and oxkMg vs position of the thymine Fig Comparison of oxki values (37 °C) for thymine oxidation in RPo–kPR and RPo–Pa complexes RPo–kPR, shadowed columns; RPo–Pa [30], open columns: in the absence of added MgCl2 (A), in the presence of 10 mM MgCl2 (B), and the Mg effect (C) 2847 Open complex at kPR promoter in the promoter sequence in relation to the transcription start point It is evident that the general pattern of relative thymine reactivity in the two complexes is similar in both the absence and presence of 10 mm Mg2+ Only the absolute values of oxk and oxkMg for thymines in RPo–kPR are two to threefold higher than those for the corresponding bases in the RPo–Pa complex This also applies to T)6, T)7 and T)10, the reactivity of which in the latter complex was too weak to permit quantitative evaluation of corresponding DNA bands in the footprints [30] This seems to be an important observation, implying higher internal dynamics of the RPo–kPR complex, associated with a larger flux of MnO4– to the reaction sites The magnitude of the Mg effect, oxki,Mg ⁄ oxki, however, is comparable in the two complexes (Fig 7C), which indicates that, in spite of some differences between the sequences of these promoters in the bubble region (Fig 1), the separated DNA strands in both complexes remain in the same protein environment The only base that exhibits different reactivity in the two complexes is T)9; in RPo–kPR in the absence of Mg2+ its reactivity is severalfold lower than that of the adjacent T)8 and apparently insensitive to temperature, whereas in RPo– Pa the two bases exhibit similar reactivity However, in both complexes, the reactivity of these bases increases similarly in the presence of Mg2+ Although in both promoters T)9 occurs in the same 3¢TAT()9) TA5¢ sequence context, at the crucial )12 position, at which the separation of DNA strands is believed to be nucleated [1,11,12], they bear different base pairs: GC in kPR and TA in Pa Thus the GC pair at this position of the )10 element apparently influences the interactions of T)9 with the protein environment The similarity, within an order of magnitude, of oxk values for thymine oxidation in free nucleotides and in RPo–kPR and RPo–Pa indicates generally high structural dynamics of the bubble region in open transcription complexes The higher dynamics of RPo–kPR than that of RPo–Pa can be related to the fact that Pa bears canonical )10 and )35 recognition hexamers, whereas in kPR they are mutated at positions )12 and )30, respectively (Fig 1) Furthermore, kPR bears from )14 to )18 position a row of five GC base pairs, whereas the whole 17-bp spacer of Pa is made up solely of AT base pairs Owing to the presence of a number of TA steps, the latter is expected to be highly flexible [39], allowing a better fit between the recognition hexamers and RNAP The greatly varied reactivity of thymines in RPo can be confronted with the current structural model of the open transcription complex [11,12] According to this model, the two DNA strands separate and take differ2848 ´ T Łozinski and K L Wierzchowski ent paths beginning at )11 bp, the upstream edge of the transcription bubble The )10 nontemplate strand element from position )11 to )7 crosses the r2 domain, where it can interact with the exposed aromatic residues of r region 2.3 (corresponding to Tyr425, Tyr430 and Trp433 of E coli r70) The low reactivity of T)6, T)7 and T)10 is thus fully consistent with the proposed stacking interactions between nucleic acid bases and the aromatic amino-acid side chains in this region [1,17,40,41]; in a stacked ‘sandwich’ state, thymine can be protected from oxidation by MnO4– either sterically or by competitive oxidation of its stacked aromatic partner, or both The low value of the Mg effect points to dielectric shielding of these bases The downstream part of the nontemplate strand, positions ˚ from )5 to )2, is held in a % 10 A wide tunnel formed by two lobes of the b (b1 and b2) subunit and the r2 domain, the 2.1 and 2.3 regions of which provide a strip of positively charged residues interacting with DNA phosphates [12] Again, the very high reactivity of T)3 and T)4 in RPo–kPR and of T)2, T)3 and T)4 in RPo–Pa, taken together with the moderate Mg effect, point to partial compensation of the negative electrostatic potential of DNA phosphates by positively charged amino acids and to a low steric barrier to the diffusion of permanganate ions to the thymines in the wide protein tunnel, in excellent agreement with the model The template DNA strand from )11 is ˚ diverted through a % 12 A tunnel completely enclosed on all sides by parts of r2, r3, b1, and the so called b¢ lid and b¢ rudder [11] The model predicts that universally conserved basic amino acids of r regions 2.4 and 3.0 (corresponding to Arg436, Lys462, Arg465 and Arg468 of E coli r70) exposed at the entrance of the tunnel, and those of region 2.2 on its wall, may contribute to the electrostatic potential responsible for stabilization of the template strand in the tunnel The high reactivity of T)11 and T)8, connected with a moderate Mg effect, is thus in good agreement with the model The path of the template strand then takes it past the r2 – r3 linker and elements of the b subunit that make up the back wall of the RNAP active-site channel, until it passes near the active site [11] The very low reactivity of the thymines close to the catalytic center region in the absence of Mg2+ associated with the large Mg effect can be interpreted as being due, for the most part, to a high electrostatic barrier to diffusion associated with the negative charges of both DNA phosphates and the carboxylic groups of three aspartates of the NADFDGD motif involved in the binding of catalytic Mg2+ ions [23,42] Chelation of these ions to the carboxylic groups may, of course, induce some local conformational changes around the FEBS Journal 272 (2005) 2838–2853 ª 2005 FEBS ´ T Łozinski and K L Wierzchowski transcription start point, also increasing steric accessibility of DNA bases to MnO4– In the light of the above considerations, the Mg-modulated reactivity of thymines reflects variation in the effective electrostatic potential along the RNAP channels holding the template and nontemplate DNA strands in the open complex The greater Mg effect on oxki in RPo–kPR at low temperatures, causing an apparent decrease in the calculated oxEa values, deserves a short comment It was not observed for the thymidine nucleotides and hence cannot be ascribed to a higher extent of diffusive binding of Mg2+ to DNA phosphates, which would lead to an increase in Kscr However, Kass for specific chelation of these ions by carboxylate groups can be expected to increase with a fall in temperature Besides aspartates in the catalytic center, these groups may occur in the protein channels in proximity to the strips of positively charged basic amino-acid residues, with which they might form salt bridges before isomerization of the complex and DNA strand separation [32] In our recent work on KMnO4 oxidation of RPo–Pa [29], we showed that Mg2+ ions exert an opposite, protective, effect on the protein part of the complex, by slowing down oxidation reactions of some amino acids in RNAP, strongly competitive with thymine oxidation in the melted DNA region In the presence of 10 mm Mg2+, the rates of RPo inactivation at °C and dissociation of strongly damaged RPo,ox complexes into components at 35 °C are twofold smaller At lower temperatures, both the expected higher occupancy of Mg-chelating sites and the smaller amplitude of breathing fluctuations in RNAP would diminish diffusion of MnO4– to vulnerable sites Simultaneous enhancement by Mg2+ of the oxidation reaction of thymines makes it more competitive, leading to higher ox ki,Mg ⁄ oxki ratios Thus, the inverse relationship between oxki,Mg ⁄ oxki and temperature, causing an apparent decrease in the corresponding activation energies of thymine oxidation, should not be considered in connection with the oxEa values determined in the absence of Mg2+ Concluding remarks Our present and earlier studies [29,30] on permanganate oxidation of RPo clearly show that careful application of this oxidant as a probe for melted DNA regions in DNA–protein complexes may yield precise information on both the position-dependent inherent reactivity of vulnerable sites in DNA and the structural dynamics of the complexes Moreover, when combined with modulation of the electrostatic barrier to diffusion of FEBS Journal 272 (2005) 2838–2853 ª 2005 FEBS Open complex at kPR promoter the anionic oxidant by Mg2+ ions, this method may also provide information on the distribution of the electrostatic potential along the single DNA strands Experimental procedures RNA polymerase RNA polymerase (EC 2.7.7.6) was prepared from E coli C600 strain as described by Burgess & Jendrisak [43] except that Sephacryl S300 was used instead of Bio-Gel A5m The enzyme was stored in buffer S (50% glycerol, 100 mm NaCl, 10 mm Tris ⁄ HCl, pH 7.9, 0.1 mm dithiothreitol) and its activity was estimated as described previously [44] at % 50% Promoters and DNA primers Promoter Pa, containing E coli consensus )35 and )10 hexamers separated by a 17-bp spacer, was synthesized, cloned into pDS3 plasmid and purified as described previously [45] The kPR promoter (an 80 bp DNA fragment from position )59 to +21 with respect to the transcription start site) was obtained by PCR using lambda phage DNA as a template and appropriately designed primers with overhanging ends encoding XhoI and EcoRI sequences The PCR product was cloned into the pDS3 plasmid, and the kPR-containing fragment purified as for Pa DNA primers used in footprinting of RPo, Pr(nt) – complementary to the nontemplate DNA strand (from +95 to +116 with respect to the transcription start site of kPR), and Pr(t) – complementary to the template DNA strand (from )159 to )138) – were synthesized by the solid-phase phosphoramidite method, purified by denaturing PAGE followed by DEAE-Sephacel column chromatography and ethanol precipitation The 5¢ ends of the primers were phosphorylated with a twofold molar excess of [32P]ATP[cP] with polynucleotide kinase All chemicals were of molecular biology or reagent grade KMnO4 oxidation Thymidine and its nucleotides The reaction was started by adding 0.1 mL freshly prepared KMnO4 solution of the desired concentration to 0.9 mL of substrate solution, 0.111 or 0.055 mm in Thd, TpT, TMP or TTP, in PSB buffer (1.11 mm potassium phosphate, pH 7.2, 111 mm KCl) at a selected [MgCl2] (0–111 mm) The final [KMnO4] of 0.625–10 mm was always high enough to ensure pseudo-first-order conditions for the oxidation reaction The reaction was terminated by adding 0.2 mL 0.2 m NaHSO3 at various times (30–120 s) depending on the temperature (1–37 °C) at which it was carried out Afterwards the solutions were spun to remove 2849 Open complex at kPR promoter traces of precipitated MnO2, and the decrease in A267, proportional to the oxidative conversion of thymine into its diglycol, was measured spectrophotometrically RPo at the Pa promoter Oxidation of thymines in RPo at the Pa promoter and detection of the oxidation products by primer extension reaction were performed as described previously [29,30] The complex, formed by incubation for 15 at 37 °C of 50-lL samples of the reaction mixture containing pmol pDS3 plasmid and pmol RNAP in TB buffer (25 mm Tris ⁄ HCl, pH 7.0, 100 mm KCl, 0.2 mm EDTA), was oxidized with a low single-hit oxidant dose, 0.01 or 0.02 m · s, at a number of MgCl2 concentrations spanning the range 0–40 mm The experiments were performed in duplicate The total intensity of the group of DNA bands corresponding to the nontemplate thymines from T)4 to T+3 was normalized to the intensity of the whole lane; both intensities were determined by volume integration using the imagequant software RPo at the kPR promoter Footprinting reactions of RPo at kPR were performed as a function of single-hit oxidant dose as described for RPo at the Pa promoter [29,30] RPo was formed as described in a recent kinetic study [6] at three selected temperatures of 13 °C, 25 °C and 37 °C using incubation times in TB buffer of 120, 60 and 30 min, respectively, and fourfold excess of RNAP KMnO4 of the desired concentration (freshly diluted from 0.1 m stock) was added, and the reaction mixture incubated for precisely at the temperature of the open complex formation Note that [KMnO4] and time of the reaction were shown to be equivalent variables of the kinetics [29] The following oxidant doses (m · s) were applied: at 37 °C )0.004, 0.008, 0.012, 0.016, 0.02; at 25 °C )0.01, 0.02, 0.03; at 13 °C )0.01, 0.02, 0.03 and 0.04 The nominal doses were corrected for the redox capacity of the buffer [29] Oxidized DNA bases were detected by the primer extension reaction using 32P-end-labeled primers Purified products of the reaction were resolved (in duplicate) on 6% polyacrylamide sequencing gels in TBE buffer (0.089 m Tris ⁄ borate, pH 8.3, and mm EDTA) Dried gels were exposed to storage phosphor screens (Molecular Dynamics, Sunnyvale, CA, USA) From each pair of gels, the one exhibiting a better resolution of bands was selected for quantitative analysis All footprinting experiments were duplicated or triplicated Phosphorimager analysis and quantification of band intensities Images of footprints were obtained with the use of a Molecular Dynamics Phosphorimager Integrated intensities 2850 ´ T Łozinski and K L Wierzchowski of bands (or groups of bands) and their intensity profiles along gel lanes were obtained with the help of the imagequant software, by volume integration and area integration of quadrilateral contours encompassing these bands, respectively For area integration, the lowest intensity point in the graph was used as the horizontal baseline (background) In volume integration, local background was used for bands corresponding to promoter bubble DNA fragments, whereas for the whole lanes that of the gel without any radioactivity was used (outside the lanes) These data were analyzed further using originä software (MicroCal Software, Northampton, MA, USA) Fractions 32Pfi of DNA fragments corresponding to oxidized T+1, T+2 and the sum of T)7 and T)10, characterized by well-separated band profiles, were determined from integrated band intensities within respective quadrilateral contours: (+2 … )1) (+3 … )1) and ()6 … )11), and normalized to that of the whole line Fractions 32Pfi of DNA fragments characterized by overlapping band profiles, corresponding to T)11, T)9, T)8 (within the )6 … )13 quadrilateral contour) and to T)4 and T)3 (within the )2 … )5 quadrilateral contour), were calculated after deconvolution of the integrated and normalized contour profiles into peaks, with use of a Lorentz distribution function, to obtain the actual band partition The primer extension reaction stops at a base preceding the oxidized thymine when the glycol form of the latter is hydrolyzed to a urea derivative during alkaline denaturation [46], which is clearly seen as band doubling for T)11 and T+2 Therefore, the measured contribution of these forms to the integrated intensity of these two bases was used in the calculation of the contribution of individual bases to the integrated intensity of overlapping DNA bands Determination of the promoter occupancy Products of the Klenow reaction can also include DNA fragments originating from unmodified plasmid molecules and ⁄ or those complexed with RNAP at sites other than the promoter Therefore, for proper interpretation of the single-exponential dependence of 32Pfi on the oxidant dose – 32P fi ¼ Fo (1-exp[–kix]) – one has to know the actual fraction Fo of promoter DNA occupied by RNAP in the form of RPo Values of Fo were determined by simultaneous fitting of the above equation to the experimental 32Pfi data (four sets for each strand at a given temperature, in the absence and presence of 10 mm MgCl2), together with the fraction of unmodified DNA: 32PfuDNA ¼ 1–32Pfbubble, where 32Pfbubble ¼ S32Pfi For the more reactive template strand, footprinted in the presence of 10 mm MgCl2, the following Fo values were obtained: 0.91 ± 0.04, 0.90 ± 0.01 and 0.81 ± 0.05, at 37 °C, 25 °C and 13 °C, respectively As the promoter occupancy should not be FEBS Journal 272 (2005) 2838–2853 ª 2005 FEBS ´ T Łozinski and K L Wierzchowski Open complex at kPR promoter smaller in a lower salt buffer, the same Fo values were assumed to represent fractions of RPo in footprinting reactions carried out in both the presence and absence of 10 mm MgCl2 Consequently, from the experimental fractions corrected for the promoter occupancy, 32Pfi ⁄ Fo ¼ RPo fi, the actual fractions of oxidized thymines in RPo were calculated as follows: ! i X ox RPo fi ẳ RPofi = fi ỵ RPo fuDNA i¼1 where RPo fuDNA ¼ À i X RPo fi i¼1 is the fraction of unmodified DNA in RPo; i numbering starts from the top of gel lanes Acknowledgements 10 The authors thank Dr Krystyna Bolewska and Teresa Rak for preparation of RNA polymerase of excellent quality 11 References Helmann JD & deHaseth PL (1999) Protein–nucleic acid interactions during open complex formation investigated by systematic alteration of protein and DNA binding partners Biochemistry 38, 5959–5967 Roe J-H, Burgess RR & Record MT Jr (1984) Kinetics and mechanism of the interaction of Escherichia coli RNA polymerase with the kPR promoter J Mol Biol 176, 495–521 Roe J-H, Burgess RR & Record MT Jr (1985) Temperature dependence of the rate constants of the Escherichia coli RNA polymerase–kPR promoter interactions Assignment of the kinetic steps corresponding to protein conformational change and DNA opening J Mol Biol 184, 441–453 Suh WC, Leirmo S & Record MT Jr (1992) Role of Mg2+ in the mechanism of formation and dissociation of open complexes between Escherichia coli RNA polymerase and the kPR promoter: kinetic evidence for a second open complex requiring Mg2+ Biochemistry 31, 7815–7825 Tsodikov OV & Record MT Jr (1999) General method of analysis of kinetic equations for multistep reversible mechanism in single-exponential regime: applications to kinetics of open complex formation between Er70 RNA polymerase and kPR promoter DNA Biophys J 78, 1320–1329 Saecker RM, McQuade LM, Schlax PE Jr, Capp MW & Record MT Jr (2002) Kinetic studies and structural models of the association of E coli RNA polymerase FEBS Journal 272 (2005) 2838–2853 ª 2005 FEBS 12 13 14 15 16 17 18 19 with the kPR promoter: large scale conformational changes in forming the kinetically significant intermediates J Mol Biol 319, 649–671 Buc H & McClure WR (1985) Kinetics of open complex formation between Escherichia coli RNA polymerase and the lacUV5 promoter Evidence for a sequential mechanism involving three steps Biochemistry 24, 2712– 2723 Rosenberg S, Kadesch TR & Chamberlin MJ (1982) Binding of Escherichia coli RNA polymerase holoenzyme to bacteriophage T7 DNA Measurements of the rate of open complex formation at T7 promoter A1 J Mol Biol 155, 31–51 Revyakin A, Ebright RH & Strick TR (2004) Promoter unwinding and promoter clearance by RNA polymerase: detection by single-molecule DNA nanomanipulation Proc Natl Acad Sci USA 101, 4776– 4780 Suh WC, Ross W & Record MT Jr (1993) Two open complexes and a requirement for Mg2+ to open the kPR transcription start site Science 259, 358–361 Murakami KS, Masuda S, Campbell EA, Muzzin O & Darst SA (2002) Structural basis of transcription initiation: an RNA polymerase holoenzyme–DNA complex Science 296, 1285–1290 Mekler V, Korthkonija E, Mukhopadhyay J, Knight J, Revyakin A, Ebright YW, Levy R & Ebright RH (2002) Structural organization of bacterial RNA polymerase holoenzyme and the RNA polymerase-promoter open complex Cell 108, 599–614 Vassylyev DG, Sekine S-I, Laptenko O, Lee J, Vassylyeva MN, Burukhov S & Yokoyama S (2002) Crystal structure of bacterial polymerase holoenzyme at ˚ 2.6 A resolution Nature 417, 712–719 Murakami KS, Masuda S & Darst SA (2002) Structural basis of transcription initiation: RNA polymerase ˚ holoenzyme at A resolution Science 296, 1280–1284 Severinov K & Darst SA (1997) A mutant RNA polmerase that forms unusual open promoter complexes Proc Natl Acad Sci USA 94, 13481–13486 Nechaev S, Chlenov M & Severinov K (2000) Dissection of two hallmarks of the promoter open complex by mutation in an RNA polymerase core subunit J Biol Chem 275, 25516–25522 Young BA, Anthony LC, Gruber TM, Arthur TM, Heyduk E, Lu CZ, Sharp MM, Heyduk T, Burgess RR & Gross CA (2001) A coiled-coil from the RNA polymerase beta¢ subunit allosterically induces selective nontemplate strand binding by sigma (70) Cell 105, 935– 944 Young BA, Gruber TM & Gross CA (2004) Minimal machinery of RNA polymerase holoenzyme sufficient for promoter melting Science 303, 1382–1384 Krakow JS, Rhodes G & Jovin TM (1976) RNA polymerase: catalytic mechanisms and inhibitors In RNA 2851 ´ T Łozinski and K L Wierzchowski Open complex at kPR promoter 20 21 22 23 24 25 26 27 28 29 30 Polymerase (Losick R & Chamberlin M, eds), pp 127– 157 Cold Spring Harbor Laboratory Press, Plainview, NY Koren R & Mildwan AS (1977) Magnetic resonance and kinetic studies of the role of the divalent cation activator of RNA polymerase from Escherichia coli Biochemistry 16, 241–249 ´ Szafranski P, Smagowicz JW & Wierzchowski KL (1985) Substrate selection by RNA polymerase from E coli The role of ribose and 5¢-triphosphate fragments, and nucleotides interaction Acta Biochim Pol 32, 329–349 Mustaev A, Kozlov M, Markovtsov V, Zaychikov E, Denissova L & Goldfarb A (1997) Modular organization of the catalytic center of RNA polymerase Proc Natl Acad Sci USA 94, 6641–6645 Sosunov V, Sosunova E, Mustaev A, Bass I, Nikiforov V & Goldfarb A (2003) Unified two-metal mechanism of RNA synthesis and degradation by RNA polymerase EMBO J 22, 2234–2244 Craig ML, Suh WC & Record MT Jr (1995) HO and DNase I probing of Er70 RNA polymerase–kPR open complexes: Mg2+ binding and its structural consequences at the transcription start site Biochemistry 34, 15624–15632 Craig ML, Tsodikov OV, MacQuade KL, Schlax PE, Saecker RM, Capp MW & Record MT Jr (1998) DNA footprints of the two kinetically significant intermediates in formation of an RNA-polymerase-promoter open complex: evidence that interactions with start site and downstream DNA induce sequential conformational changes in polymerase and DNA J Mol Biol 283, 741– 756 Zaychikov E, Denissova L, Meier T, Gotte M & Heumann H (1997) Influence of Mg2+ and temperature on formation of the transcription bubble J Biol Chem 272, 2259–2267 Chen Y-F & Helmann JD (1997) DNA melting at the Bacillus subtilis flagellin promoter nucleates near )10 and expands unidirectionally J Mol Biol 267, 47–59 Tsodikov OV, Craig ML, Saecker RM & Record MT Jr (1998) Quantitative analysis of multiple-hit footprinting studies to characterize DNA conformational changes in protein-DNA complexes: application to DNA opening by Er70 RNA polymerase J Mol Biol 283, 757–769 ´ Łozinski T & Wierzchowski KL (2003) Inactivation and destruction by KMnO4 of Escherichia coli RNA polymerase open transcription complex: recommendations for footprinting experiments Anal Biochem 320, 239– 251 ´ Łozinski T & Wierzchowski KL (2001) Mg2+ ions 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 2852 31 32 33 34 35 36 37 38 39 40 41 42 43 quantitative KMnO4 footprinting study Acta Biochim Pol 48, 495–510 ´ Kolasa IK, Łozinski T & Wierzchowski KL (2001) Mg2+ does not induce isomerization of the open transcription complex of Escherichia coli RNA polymerase at the model Pa promoter bearing consensus )10 and –35 hexamers Acta Biochim Pol 48, 985–994 Saecker RM & Record MT Jr (2002) Protein surface salt bridges and paths for DNA wrapping Curr Opin Struct Biol 12, 311–319 ´ Łozinski T & Wierzchowski KL (2001) Effect of Mg2+ on kinetics of oxidation of pyrimidines in duplex DNA by potassium permanganate Acta Biochim Pol 48, 511– 523 Hayatsu H & Ukita T (1967) The selective degradation of pyrimidines in nucleic acids by permanganate oxidation Biophys Biochem Res Commun 29, 556–561 Saenger W (1984) Principles of Nucleic Acid Structure Springer Verlag, New York ´ Poznanski J, Felczak K, Kulikowski T & Remin M (2000) 1H NMR conformational study of antiherpetic C-substituted 2¢ deoxyuridines: insight into the nature of structure–activity relationships Biochem Biophys Res Commun 272, 64–74 Walaas E (1958) Stability constants of metal complexes with mononucleotides Acta Chem Scand 12, 528–536 Strainic MG Jr, Sullivan JJ, Velevis A & deHaseth PL (1998) Promoter recognition by Escherichia coli RNA polymerase: Effects of the UP element on open complex formation and promoter clearance Biochemistry 37, 18074–18080 ´ Kolasa IK, Łozinski T & Wierzchowski KL (2003) Effects of distortions by A-tracts of promoter B-DNA spacer region on the kinetics of open complex formation by Escherichia coli RNA polymerase Acta Biochim Pol 50, 909–920 Juang Y-L & Helmann JD (1994) A promoter melting region in the primary sigma factor of Bacillus subtilis Identification of functionally important aromatic amino acids J Mol Biol 235, 1470–1488 Malhotra A, Severinova E & Darst SA (1996) Crystal structure of a r70 subunit fragment from E coli RNA polymerase Cell 87, 127–136 Zaychikov E, Martin E, Denissova L, Kozlov M, Markovtsov V, Kashlev M, Heumann H, Nikiforov V, Goldfarb A & Mustaev A (1996) Mapping of catalytic residues in the RNA polymerase active center Science 273, 107–109 Burgess RR & Jendrisak JJ (1975) A procedure for the rapid, large scale purification of Escherichia coli DNAdependent RNA polymerase involving polyamine P precipitation and DNA-cellulose chromatography Biochemistry 14, 4634–4638 FEBS Journal 272 (2005) 2838–2853 ª 2005 FEBS ´ T Łozinski and K L Wierzchowski 44 Chamberlin M, Kingstone R, Gilman M, Wiggs J & deVera A (1983) Isolation of bacterial and bacteriophage RNA polymerases and their use in synthesis of RNA in vitro Methods Enzymol 101, 40–568 ´ 45 Łozinski T, Adrych-Rozek K, Markiewicz WT & Wierzchowski KL (1991) Effect of DNA bending in various regions of a consensus-like Escherichia coli promoter on FEBS Journal 272 (2005) 2838–2853 ª 2005 FEBS Open complex at kPR promoter its strength in vivo and structure of the open complex in vitro Nucleic Acids Res 19, 2947–2953 46 Ide H, Kow YW & Wallace SS (1985) Thymine glycols and urea residues in M13 DNA constitute replicative blocks in vitro Nucleic Acids Res 13, 8035– 8052 2853 ... 0.25 0.05 1.7 of the t strand The patterns of thymine reactivity in the presence and absence of Mg2+ were generally similar In the absence of Mg2+, the reactivity of all the thymines was merely... sequences of these promoters in the bubble region (Fig 1), the separated DNA strands in both complexes remain in the same protein environment The only base that exhibits different reactivity in the. .. thymines reflects variation in the effective electrostatic potential along the RNAP channels holding the template and nontemplate DNA strands in the open complex The greater Mg effect on oxki in RPo–kPR