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 cond
Trang 1transcription complex reflects variation in the negative electrostatic potential along the separated DNA strands
Footprinting of Escherichia coli RNA polymerase complex
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; kP R
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: klw@ibb.waw.pl
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 (13–37C) 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 oxkproved 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
oxkwith 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 (13–37C) 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.
Trang 2open complex (RPo) capable of specific binding of
NTP substrates and synthesis of nascent RNA [1]
Kinetic–mechanistic studies on the initiation of
tran-scription 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
ini-tial complex (called I1 or RPc) and an intermediate
one (I2or RPi):
Rþ P !k1
k 1
I1 !k2
k 2
I2 !k3
k 3
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
(K3¼ k3⁄ k-3) towards RPo [3,7,9] For the kPR
pro-moter on a negatively supercoiled plasmid, this
equilib-rium has been found shifted completely towards RPo
over the temperature range 4–37C [10]
According to the current molecular model of RPo
[11,12], based on crystal structures of Thermus
thermo-philus [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
mole-cular 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
inter-mediates [15,16], identification of RNA polymerase
(RNAP) regions involved in melting, and
demonstra-tion 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
pro-cess 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
for-mation and binding of NTP substrates [20–23], a
pos-sible role of these ions in RPo formation has been
probed by kinetic [4] and footprinting experiments [10,24–26] A large difference between the observed ( 0.4) and expected ( 4; from that of 7 found for
Na+[2]) stoichiometry of Mg2+ion uptake in the kin-etically 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 tran-scription-competent form, RPo2 Strong enhancement
by Mg2+ of susceptibility to KMnO4 oxidation of pyrimidine bases at the )12, +1 and +2 positions in
kPRpromoter 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 resi-dues 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 asso-ciated 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, indi-cated 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 melt-ing 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 permangan-ate 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 indi-vidual bases Application of this method to perman-ganate oxidation of RPo–kPRshowed that of the three bases (T)4, T)3 and T+2) probed at 37 C and 0 C, only the reactivity of the last one was
Trang 3temperature-dependent [28] Our attempts to apply this method to
RPo at a synthetic Pa promoter failed, however,
because of the occurrence of highly competitive
oxida-tion reacoxida-tions within the RNAP component [29], which
was ignored in the method We have shown that under
multiple-hit KMnO4 doses commonly used in the
ear-lier footprinting experiments, RPo becomes completely
inactivated, through severe damage by multiple
oxida-tive lesions accumulating in both the RNAP and the
melted DNA region, and partially dissociated
Perman-ganate footprinting of RPo–Pa as a function of
single-hit oxidant dose [30] showed that, in this complex,
Mg2+ions do 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
com-plex, 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 20–37C temperature range,
four Mg2+ions are involved in the equilibrium K3¼
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
dis-sociation show that its course is biphasic, which may
indicate involvement of ionic exchange coupled to
re-formation 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
foot-printing 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
per-manganate 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 37C is generally similar to
that determined previously by us for RPo at the Pa
promoter on the same template under similar
condi-tions [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
More-over, 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
oxi-dation in RPo–Pa [30] and in dsDNA [33] was due
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 dithymi-dine (3¢-5¢)-monophosphate (TpT) as a function of temperature and [Mg2+]
In connection with the recent model of RPo struc-ture [11,12], we show finally that the observed reacti-vity 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–kPRcomplex Thymine residues in the promoter bubble DNA region of RPo formed by E coli RNAP at the kPR
promoter (see Fig 1 for sequence), contained in the pDS3 plasmid, were oxidized by KMnO4 in the absence and presence of 10 mm MgCl2 at three selec-ted temperatures (13C, 25 C and 37 C) as a func-tion of the oxidant dose (x¼ [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 pro-tein fragments forming this site [22] PAGE-resolved DNA products of the Klenow extension reaction,
Fig 1 Sequences of the kP R and Pa promoters studied Melted regions in RPo are shown in bold, )10 and )35 recognition hexa-mers are underlined, and an arrow marks the transcription start point.
Trang 4corresponding to oxidized thymine, are exemplified in
Fig 2 Inspection of the gels shows that DNA bands
corresponding to all thymines in the )11 … +2
pro-moter region came up clearly in the footprints
obtained even at 13C and the lowest oxidant dose
applied and in the absence of MgCl2 In the
foot-prints 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
pre-sent 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
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 mono-exponentially as a function of the applied oxidant dose
x, as expected for a pseudo-first-order reaction This is exemplified in Fig 3 for reactions performed at 37C The corresponding rate constants of the reaction, oxki,
A
B
Fig 2 Selected KMnO 4 footprints of the melted DNA region of RPo at the kP R 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 32 P-end-labeled ssDNA products of the Klenow primer extension reaction carried out on the nontemplate (A) and template (B) DNA strands; doses of KMnO 4 (in M · s) applied at 37 C are indicated above lanes 2–11 Minus and plus signs indicate the absence and pres-ence of 10 m M MgCl2in footprinting reactions Lane 1, footprint of dsDNA without RNA polymerase (C) and (D) Fragments of autoradio-grams of 6% polyacrylamide sequencing gels showing resolved 32 P-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 KMnO 4 doses (indicated above the lanes); along the leftmost lanes positions of DNA bands corresponding to thymines in the melted region of the kP R 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.
Trang 5were thus obtained by nonlinear weighted fit of Eqn
(1) to the experimental data:
ox
fi¼ 1 expðoxki xÞ ð1Þ They are collected in Table 1 The pseudo-first-order
character of the oxidation reaction testifies that all
thy-mines 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
At 37C 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
Fig 3 Kinetics of KMnO 4 oxidation at 37 C
of thymines in the melted region of RPo at the kPRpromoter Data points (mean val-ues: n ¼ 3, calculated standard errors in the range of 10–15%) corresponding to DNA fractions of oxidized thymines ( ox fi) and unoxidized DNA (f uDNA ) in the template (left column), and nontemplate (right column) strands, in the absence (j) and presence (d) of 10 m M MgCl2were obtained by quan-tification of the footprints (exemplified in Fig 2) as described in Experimental Proce-dures; solid lines represent fitted functions (Eqn 1,oxk i values in Table 1).
Trang 6of 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 37C
to 25C and 13 C; only the rate constants of oxida-tion became progressively smaller, as would be expec-ted 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
1⁄ T (K) were linear (Fig 4, correlation coefficient )0.98 or better) as ifoxkireflected mainly the tempera-ture dependence of the oxidation reaction The ener-gies of activation, oxEa, calculated from these plots (Table 2) for reactions carried out in the absence of
Mg2+ were 6–8 kcalÆmol)1 (25.1–33.5 kJÆmol)1) for T)3, T)4, T)7, T)10 and T)11, distinctly higher at
11 kcalÆmol)1 ( 46 kJÆmol)1) for T+2 and T+1, and much lower at 3.7 kcalÆmol)1 (15.5 kJÆmol)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 pre-sence 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+
Table 1 Pseudo-first-order rate constants ( ox ki) for thymine
oxida-tion by KMnO4in the melted DNA region of RPo at the kPR
promo-ter The mean ± SEM values of oxk i were determined from
nonlinear weighted least squares fit of Eqn (1) to the ox fi(x) data
(Fig 3) obtained as described in Experimental procedures nt,
Non-template DNA strands; t, Non-template DNA strand.
Thymine
Temperature
(C)
ox k i ( M )1Æs)1)
[Mg2+] ¼ 0
ox k i,Mg ( M )1Æs)1)
[Mg2+] ¼ 10 m M oxk i,Mg ⁄ ox
k i
T-7 &
T-10 (nt)
0.8 ± 0.1 1.0 ± 0.1 1.25 ± 0.1
T-7 &
T-10 (nt)
0.5 ± 0.1 0.9 ± 0.1 1.8 ± 0.4
T-7 &
T-10 (nt)
0.3 ± 0.1 0.6 ± 0.1 2.0 ± 0.75
Fig 4 Effect of temperature and Mg 2+ on the rate constants of thymine oxidation by KMnO4in RPo at the kPRpromoter, in Thd and TMP Arrhenius plots of ox kidata (Tables 1 and 3) in the absence (j) and presence (d) of 10 m M MgCl2; solid lines represent fitted functions, cor-responding activation energies of the reaction in Table 2.
Trang 7There 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
regar-ded 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
Discus-sion
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
pres-ence of 100 mm KCl (Figure 5 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 1 and
2) For the nucleotides, they were smaller than for the
parent nucleoside (21.1 m)1Æs)1) and decreased with the
increase in negative charge on the phosphate group in
the order TpT (14.4 m)1Æs)1), TMP (8.5 m)1Æs)1) and
TTP (6.4 m)1Æs)1)
In agreement with the hypothesis referred to above,
the rate of Thd oxidation was independent of the
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
Table 2 Energies of activation of the KMnO4-oxidation reaction
( ox Ea) of thymines in the melted DNA region in RPo at the kPR
pro-moter, and in Thd and TMP The mean ± SEM values ofoxE a were
determined from linear weighted least-squares fit of the Arrhenius
equation to the ox kidata from Table 1 and Table 3.
Substrate
ox Ea
(kcalÆmol)1)
[Mg2+] ¼ 0
ox Ea,Mg (kcalÆmol)1) [Mg2+] ¼ 10 m M oxE a,Mg ⁄ ox
E a
T-7 & T-10 (nt) 7.2 ± 0.1 3.0 ± 1.7 0.42 ± 0.24
[Mg 2+ ] ¼ 50 m M
8.3 ± 0.1
Fig 5 Effect of [MgCl 2 ] 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 constantsoxk of the reaction in nucleo-tides were determined at 25 C, and sums of the respective ox kiin the RPo–Pa complex at 37 C Solid lines represent fitted functions (Eqn 2), the values of the fitted parameters in Table 4.
Trang 8bond [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
tri-phosphate 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
oxk([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
kMg¼oxkþ Df½Mg2þ Kscr=ð1 þ ½Mg2þ KscrÞg
ð2Þ where Kscr is a screening constant, expected to be
pro-portional to the corresponding thermodynamic binding
constant, Kass, for Mg2+ and D ¼oxk([Mg]fi 1) –
oxk([Mg]¼ 0) is an increment by which the initial value
ofoxk would increase at the saturating Mg2+
concen-tration Values of Kscr thus obtained at 25C (25.7,
32.4 and 350 m)1) for TpT, TMP and TTP,
respect-ively (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
screen-ing 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
adop-ted by the Mg-chelating triphosphate group [35], some
of which apparently do not contribute significantly to
the electrostatic barrier to MnO4 being considered
The corresponding values of oxk([Mg] fi 1)¼ D +
oxk([Mg] ¼ 0), that is the rate constant at saturating
Mg2+concentration (18.3, 15.5 and 13.5 m)1Æs)1) tend
to approach that of 21.1 m)1Æs)1 measured for thymi-dine 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
oxk([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 stack-ing of thymine residues brstack-ings the two C(5)¼C(6) bonds in close proximity, thereby increasing the prob-ability of their attack by MnO4 and decreasing the effect of the negative phosphate charges on MnO4 dif-fusion by dielectric shielding
The temperature dependence of the kinetics of oxi-dation 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+
Table 3 Pseudo-first-order rate constants ( ox k) for thymine oxidation by KMnO4in Thd and its nucleotides The mean ± SEM values of ox k were determined from nonlinear weighted least-squares fit of a single exponential decay function to the kinetic data obtained as described
in Experimental Procedures.
Compound
Temperature (C)
ox k ( M )1Æs)1)
[Mg 2+ ] ¼ 0
ox k Mg ( M )1Æs)1)
[Mg 2+ ] ¼ 10 m M
ox k Mg ( M )1Æs)1)
[Mg 2+ ] ¼ 50 m M
Table 4 Fitted parameters of Mg2+-screening isotherms for KMnO4oxidation 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 5.
Compound
Temperature (C)
K scr
( M )1)
ox
k ([Mg] ¼ 0)
( M )1Æs)1)
ox
k ([Mg] fi 1)
( M )1Æs)1)
Trang 9concentrations (Table 2 and Fig 4) They are of the
same order of magnitude as those determined for
thy-mine 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
phos-phate group, thereby diminishing the electrostatic
bar-rier 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
environ-ments 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 37C exhibits a very
sim-ilar 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 37C with a KMnO4 dose of 0.01 or
0.02 m· s, found to be sufficiently low to secure
single-hit conditions of oxidation within the whole range of
MgCl2 concentrations applied [29] The corresponding
footprints (Fig 6) were quantified, and the kinetic
parameters derived as described for the RPo–kPR com-plex 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 5 The plots resemble closely the screen-ing isotherms obtained for the nucleotides Fittscreen-ing 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 sim-ilar 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 Con-sequently, the maximum value ofSoxki([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 reactiv-ity 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 com-pletely inactivated transcriptionally [29],
conformation-al changes in the catconformation-alytic 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 conclu-sion also applies to RPo at kPRand 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
Fig 6 Effect of [MgCl2] on KMnO4footprint of the melted DNA region in RPo at the Pa promoter Autoradiogram of polyacrylamide sequen-cing gel (6%) showing resolved 32 P-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; [MgCl 2 ] in m M is indicated above the lanes; along the leftmost lane positions of DNA bands corresponding to thymines in the melted region are indicated.
Trang 10promoter are susceptible to permanganate oxidation in
the temperature range 13–37C and (b) the
corres-ponding reaction rate constants are of the same order
of magnitude as, and exhibit similar Mg2+ and
tem-perature 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–kPRand 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 do 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
stopped-flow spectrofluorimetric investigations of the kinetics
of RPo formation at a synthetic promoter bearing
con-sensus )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
pro-cess, there are no intermediates with lifetimes longer
than 1 s
The normal temperature-dependence of the thymine
oxidation reaction demonstrated in RPo calls into
question the results of earlier footprinting
investiga-tions using permanganate and⁄ or other chemical
probes, in which temperature was used to visualize the
allegedly stepwise opening of stable transcription
com-plexes under the assumption that the underlying
chemi-cal reaction is temperature-independent [10,24–28]
Investigations of thymine oxidation in Thd and its
nucleotides in solution have unequivocally
demonstra-ted 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
thymine oxidation in the absence of Mg2+ between nucleotides bearing variously charged and sized phos-phate 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 barri-ers to this process The general similarity of the kinetic characteristics of the oxidation reaction in the nucleo-tide and RPo–kPRand RPo–Pa systems allows analog-ous interpretation of the observed differences in the rate constants for individual thymines in RPo in terms
of the electrostatic and steric barriers to MnO4 diffu-sion 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–kPRand RPo–Pa complexes determined under the same experimental conditions This comparison is shown in Fig 7 as col-umn plots of oxkandoxkMgvs position of the thymine
Fig 7 Comparison of ox k i values (37 C) for thymine oxidation in RPo–kP R and RPo–Pa complexes RPo–kP R , shadowed columns; RPo–Pa [30], open columns: in the absence of added MgCl2(A), in the presence of 10 m M MgCl 2 (B), and the Mg effect (C).