Download SUMMARY The steady state kinetics of initiation of T7 DNA transcrip

Volume 4 Number 11 November 1977
Nucleic AcilJS Research
Steady state kinetic studies of initiation of RNA synthesis on T7 DNA in the presence of
rifampicin
J.W.Smagowicz and K.H.Scheit
Max-Planck-lnstitut fur Biophysikalische Chemie, Abteilung Molekulare Biologie, D-3400
Gottingen, GFR
Received 19 August 1977
SUMMARY
The steady state kinetics of initiation of T7 DNA transcription by RNA polymerase holo enzyme from.E.coli in the presence
of rifampicin and the two substrates ATP and UTP were studied.
Under these conditions, the enzyme catalyzes exclusively the
promotor specific synthesis of pppApU. The kinetic data are in
agreement with the mechanism of a truly ordered reaction. Binding
of the initiating nucleotide ATP to the transcriptional complex
occurs prior to the binding of the substrate UTP. Release of
pppApU ist most probable the rate limiting step. K^, constants
were found to be 0.6 mM for ATP and 0.31 mM for UTP, respectively. The substrate inhibition pattern indicated that the substrate
site exhibits a finite affinity for incorrect nucleoside triphosphate (K-L = 2.3 mM) . A similar non specific binding to the 3-OH
site could not be demonstrated.
INTRODUCTION
Transcription of T7 DNA by RNA polymerase holo enzyme from
E.coli is specifically initiated at three promotors. The first
event in the synthesis of a RNA molecule is the binding of two
nucleoside triphosphates possessing bases complementary to a specific sequence of the promotor, followed by the formation of a
dinucleoside tetraphosphate pppXpY with concomitant release of
pyrophosphate. In accordance with this, the transcriptional complex is supposed to have an initiation site for the binding of
the initiating nucleoside triphosphate and an elongation site for
the binding of the selected substrate . An attempt to analyze the
kinetics of initiation of RNA synthesis on T7 DNA was undertaken
by Rhodes and Chamberlin , employing the so called rifampicin
challenge assay . Their results led these authors to the conclusion that initiation follows a mechanism, in which the second
nucleoside triphosphate associates rapidly with the T7 DNA enzyme
complex, followed by the binding of the initiating nucleoside
O Information Retrieval Limited 1 Falconberg Court London W1V5FG England
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triphosphate in a slow reaction, rate limiting for the initiation
process. Rhodes and Chamberlin assumed that two competing reactions occur in the presence of rifampicin: (1) chain initiation
and (2) inactivation by rifampicin, governed by the rate of rifampicin attack on the T7 DNA enzyme complex. It was, however,
shown by Johnston and McClure
that the ternary complex T7 DNA
enzyme rifampicin still catalyzes the synthesis of the initiating
pppXpY. Furthermore, Rhodes and Charaberlin measured the rate of
32
Y-( P)XTP incorporation by acid precipitation. Because only RNA
chains from a minimal chainlength on can be precipitated by acid,
this technique did not allow to directly determine the effect of
substrate concentration on the primary event of initiation. As
observed recently, the promotor specific synthesis of pppApU is
the only reaction carried out by RNA polymerase on T7 DNA in the
presence of sufficiently high concentrations of rifampicin and
ATP and UTP as substrates . The enzyme does not dissociate from
the template after release of pppApU and reaction proceeds until
exhaustion of substrates. As already pointed out by Johnston and
McClure, this reaction should be highly suitable for steady state
kinetic studies of promotor specific initiation of RNA synthesis.
In this paper we report steady state kinetic studies of initiation on T7 DNA by RNA polymerase from E.coli in presence of rifampicin. On the basis of the obtained kinetic data, we attempted
to differentiate between possible mechanisms of initiation.
MATERIAL AND METHODS
Enzyme
RNA polymerase holo enzyme from E.coli has been purified
according to Zillig
through the DEAE cellulose step and then by
affinity chromatography on heparin-sepharose as described by
Sternbach et al. . The isolated holo enzyme had a specific activity of 16.OOO units/mg assayed with T7 DNA and contained one
equivalent of sigma subunit, as determined by the rifampicin
challenge assay . The polymerase preparation was judged 95% pure
by SDS-polyacrylamide gel electrophoreses. The enzyme was essentially free from phosphatase, RNase and DNase activities in de14
terminations with 4-umbe]
4-umbelliferylphosphate, ( C)poly(rU) and
phage DNA as substrates.
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Chemicals
ATP and UTP were purchased from Boehringer
(Mannhaim),
( 14 C)UTP (sp.act. 486 mCi/mmole) and Y ~ ( 3 2 P ) U T P from AmershamBuchler (Braunschweig). The chemical purity of ATP and UTP was
greater than 95%. Contaminations of both substrates by other
nucleotides, which might have effected the experiments described
below, could not be detected. T7 DNA was isolated from purified
phage as described in the literature
and shown to be essentially
free of single-strand breaks by analytical ultracentrifugation.
Conditions for synthesis of pppApU
The enzyme-DNA complex was prepared in a total volume of 50 pi
by adding 10 pmoles of holo enzyme and 10 pmoles T7 DNA to buffer
containing: 0.04 M Tris-HCl, pH 7.9, 0.05 M NaCl, O.O1 mM EDTA,
0.01 MgCl2 and 1 mM mercaptoethanol. After 10 minutes at 37°,
rifampicin was added to a final concentration of 10 yM, followed
by further incubation for 10 minutes. The reaction was started
by adding ATP and UTP in concentrations as indicated in figures
and it was stopped after 30 minutes. The rate of synthesis was
linear for at least 90 minutes. Aliquots of the reaction mixture
4
14
were chromatographed on paper as described when ( C)UTP was
employed. The chromatogram was cut into strips, which were
measured for radioactivity in toluene based scintillation fluid.
When y-(
P)UTP was used, the rate of synthesis was determined
32
by measuring the release of ( P)-pyrophosphate as described in
Q
the literature . The structural proof of the pppApU synthesized
was carried out according to Johnston and McClure .
RESULTS AND DISCUSSION
Enzymatic condensation of two nucleoside triphosphates giving
a dinucleoside tetraphosphate with release of pyrophosphate represents a B bi-bi"reaction, e.g. the process is bimolecular in
both directions. With respect to the mode of substrate binding
and product release, the reaction may be thermodynamically random or ordered. Randomness would imply that binding of ATP is
possible either before or after UTP binding as well as release
of PP^
ma
Y proceed either before or after release of pppApU. In
a truly ordered reaction the temporal sequence of addition of
substrates and release of products is obligatory. In a random
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I
T
UTp. const.
4
-8
/
.085 rrM
3
y
2
/
/
.118rrM
^
.164 nW
/o
1
^^-—
j ^ .595rr*1
"^^^__^___..80rnM
'
-—Q
p-
o
5
3.87 mM
10
15
VATP mM"1
Fig. 1: Effect of ATP on the rate of pppApU synthesis
»
20
30
W
m
M
Fig. 2: Effect of UTP on the rate of pppApU synthesis
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schema it is possible that one of the routes if favored under
normal conditions, but all of them are principally possible.
This would be called a kinetically ordered mechanism. One could
also think about "mixed-type" mechanisms, in which substrates
bind randomly, while product release is ordered or vice versa.
The steady state kinetic studies presented here, do not provide
any information about mode and sequence of product release. Detailed product inhibition studies would clarify this point. Thus,
in the following discussion, we restrict ourselves to the mechanism of substrate binding.
The kinetic studies furnished the following experimental results:
(1) The plots 1/v versus 1/s are linear over a range of concentration from 0.05 mM to 1 mM for both ATP and UTP, respectively
(Figs. 1 and 2 ) .
(2) The slope of the plot 1/v versus 1/ATP becomes zero for saturating concentrations of cosubstrate in contrast to the slope of
the plot 1/v versus 1/UTP
(Figs. 3 and 4 ) .
(3) At high concentrations of ATP (> 1 m M ) , the rate of synthesis
decreased with increasing concentrations of ATP. The respective
double reciprocal plot is curved upwards (Fig. 5 ) . This was not
observed for high concentrations of UTP. The plot 1/v versus
1/UTP is linear up to UTP concentrations of 4 mM.
(4) The steady state kinetic studies yielded the following kinetic parameters/ &,.__ = 0.7 mM, Kj,_p = 0.31 mM and turnover number = 30/minute (Fig. 6) assuming that all enzyme molecules were
active. A plausible mechanism for the synthesis of pppApU must be
in agreement with the experimental results summarized above.
Three possible models of substrate binding are presented below:
random, ordered and kinetically ordered. Their rate equations are
given and discussed in conjunction with the experimental data.
The possible models describing the synthesis of pppApU presented below share one common feature. Based on the fact that the
Q
turnover number for elongation
was reported to be 60 times high-
er than that determined for the synthesis of pppApU and assuming
that in first approximation the rates of phosphodieser bond formation in initiation and elongation should be similar, the catalytic step in the synthesis of pppApU should not be rate limiting.
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15
ATP
VATP
mM~
Fig. 3: Replot of slopes i/rjTp- Data are taken from Fig. 2.
1
X
£.2
3
/
/o
.1
Kurp= 0.27 nH
2
6
10
VUTP TM'1
Fig. 4: Replot of slopes - j / A T p • Data are taken from Fig. 1
Model 1: Random mode of substrate binding
A
U
k-c
•pppApU + PPj
EU
U
Scheme 1
Scheme 1 gives a general mechanism of the reaction in Cleland's notation where E represents open (or rapidly starting)
enzyme-DNA complex, the rate equation can be obtained using the
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King-Altman procedure
i [A] 2 + j [A]
v
(D
V
max
k + 1 [A]2 + m [A]
where i = K1 [U], j = K 2 [U] + K 3 [U] 2 , k = K 4 + KgtU] + Kg[U] 2 ,
I = K? + Kg[U], m = Kg + K 1Q [U] + K ^ C U ] 2 .
All constants represent linear combinations of rate constants of
elementary steps. It is clear from an inspection of equation (1)
that the above rate equation, containing higher powers of substrate concentration, does not give neither linear double reciprocal plots nor replots of slopes. If rapid or partial rapid
bimolecular substrate association is assumed, the rate equation
for scheme 1 becomes:
v
Const.. [A] [U]
1
V
max
+ aKA [U] + aKy [A] + Const2 [A] [U]
(2)
For true equilibrium: K. and IC. are true dissociation constants
for ATP and UTP (KA = k_1/k1; Ky = k_ 2 /k 2 ); a is a factor by
which the dissociation constant is changed upon binding of the
other substrate (a = k_,k2/k,k_2 = k_^k-/kijk_^) and Const^ =
Const2 = 1. For partial equilibrium, all K's of equation (2) are
dynamic constants, as in the general rate equation (1). The
slopes of plots 1/v (reciprocal rates) versus 1/s (reciprocal
substrate concentration) are given by:
sloPe 1 / U T p = C o n 5 ^ V
i
slo P e 1/ATp =c o n s ^
nicLX
v
I max
M +V[U])
(4
For saturating concentrations of cosubstrate one obtains
^ V
* °
<
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Although equation (2) predicts linearity for plots 1/v versus
1/s, both replots of slopes should not go through zero at
saturating concentrations of cosubstrate, in clear contradiction to the experimental results. Thus scheme 1 cannot represent
the relevant mechanism of reaction.
Model 2: Ordered mode of substrate binding
pppApU+pp.
Scheme 2
The rate equation for scheme 2 is of the form:
v
[A] [U]
(7)
^max
K
K
iA mU
+ K
mU
[A] + K
mA
[U]
kok
where
Inspection shows that equation (7) is similar to that of
scheme 1, the only difference is that the constants represent
different combinations of rates of elementary steps. Also in
this case neither of the slope replots should go through zero
for saturating concentrations of cosubstrate. Rapid equilibrium
is attained, if the rate of product release (k, in scheme 2) is
much smaller than all other rate constants in scheme 2. With
this assumption K ^ of equation (7) can be neglected. The same
holds, if partial rapid equilibrium is assumed, e.g. if dissociation of the first substrate (k ,) is much faster than other
rates. This is equivalent to the condition: K.^ >> K .. Thus,
for rapid or partially rapid equilibrium, rate equation (7) reduces to:
v
[A] [U]
(8)
max
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K
iAISnU
+
K
mU[Al
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3
Fig.
VxTP mM"'
5: Effect of high substrate concentrations on the rate of
pppApU synthesis, (o-o-o), variable ATP concentration;
(D-O-o), variable UTP concentration.
Fig.
6: Replots of intercepts at ordinate. Data are taken from
Fig.
1.
Slopes of plots 1/v versus 1/s are given by the following
equations:
K
slope 1/ATP
iA 'SnU
d/K^/EA])
slope 1/UTP
(9)
(10)
max
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For saturating concentrations of cosubstrate holds:
=
K
mU / V max
If UTP is the first substrate to be added, then the term K p[A]
in equation (8) is substituted by K ^ [ U ]
lim slope,,
* 0 (13)
[A]-«o
VUTP
and
lim slope .
+ 0
[U]-«°
1/ATP
(14)
Replot of slopes, shown in Figs. 3 and 4, clearly indicate that
the experimental data are consistant with the model of an ordered
reaction, in which ATP binds to the transcriptional complex before UTP. To decide whether this sequence is obligatory or is
due to kinetic reasons, we considered an additional model.
Model 3: Kinetically ordered substrate binding
The rate equation and the reaction scheme for model 3 are
identical with those of model 1. Non linear plots 1/v versus 1/s
should be observed. In spite of the complexity of rate equation
(1) , on the basis of qualitative arguments it can be predicted
that deviations from linearity should be expected for plots 1/v
versus 1/UTP concentrations. Let us assume that the experimentally determined sequence of substrate binding is due to the
fact that the reaction path E + A + EA + U + E (AU) to the central complex, in the normal range of concentrations if favored
over the alternative reaction path E + U + E U + A + E (AU), or to
say k-j-kj
>>
^ 2 ^ 4 ^n
scneme
1•
At
higher concentrations of UTP,
the kinetic factors favoring the first pathway should be overcome
and larger partions of the reaction flux would proceed via the
alternative pathway. The initial rate should pass through a maximum and then decrease to a limiting plateau value, governed by
the rate constants of the kinetically less favored reaction path.
This would correspond to a minimum in the plot 1/v versus 1/UTP.
The data shown in Fig. 5, however, do not show a minimum in the
plot 1/v versus 1/UTP at high UTP concentrations.
The results of the steady state kinetic studies of pppApU synthesis are thus in agreement with a kinetic scheme of a truly
ordered substrate binding (model 2 ) . Prequisite for the binding
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of the substrate UTP is the presence of the initiating nucleo9ide
triphosphate ATP on the transcriptional complex. For such a mechanism, a higher affinity of UTP than of ATP to the transcriptional complex, as experimentally observed, should be expected.
Substrate inhibition
The deviation from linearity of the plot 1/v versus 1/ATP
(see Fig. 5) can be interpreted on the basis of the known unspecific binding of substrates with non complementary bases to the
transcriptional complex. If ATP interacts with EA then the "dead
end" complex E(A,A) is formed and characteristic substrate inhibition occurs. The rate equation for this type of inhibition
is of the form:
1
1
v
[A]
1
V
max
r
K
uI
mU
[U]
[A]
K,
i
1
(15)
JJ
where K^ is the inhibition constant for an incorrect substrate.
Inspection of equation (15) shows that the plot 1/v versus 1/ATP
had to be non-linear. Differentiation of equation (15) by 1/ATP
and setting the derivative equal to zero gives the condition for
the minimum of the plot 1/v versus 1/ATP in Fig. 5.
(16)
which for the equilibrium case reduces to
[A]
min
=
Substituting the experimental values [ATP] .
= 1.25 mM and
K ^ = 0.7 mM into equation (17) , one obtains K ± = 2.3 mM. This
value is in good agreement with previously determined inhibition
constants for incorrect substrate binding to the elongation
site
. There is no inhibition of synthesis by high concentra-
tions of UTP (Fig. 5 ) . This indicates that the 3'-OH site of the
transcriptional complex displays no affinity for nucleotides
with non complementary bases or at least that this affinity is
much lower than that of an incorrect substrate for the substrate
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site of the transcriptional complex.
Encouraged by the results of this kinetic study, we wish to
advance the following thoughts concerning the structural organization of the transcriptional complex. We assume that the principal structural features of the transcriptional complex in the
initiation and elongation mode are identical. The conditio sine
qua non is always the existence of a nucleotide, either the
initiating nucleoside triphosphate or the terminal nucleotide of
the growing RNA chain, at the 3'-OH
(or primer) site of the
transcriptional complex. The binding of substrate occurs, followed by phosphorylation of either 3'-OH terminus of the growing
RNA chain or 3'-OH of the initiating nucleoside triphosphate. It
is a priori not obvious why the transcriptional complex should
not bind initiating nucleoside triphosphate and substrate randomly,
if primer site and substrate site exist simultaneously. The
simplest explanation is that the substrate site (or elongation
site) does not exist until the binding of the initiating nucleoside triphosphate. This explains why UTP did not interfere
with the binding of ATP in the synthesis of pppApU without invoking an exclusive affinity of the primer site at the enzyme for
purine nucleoside triphosphates. The primer site has to accomodate any of the four nucleotides if the template sequence demands this. In this context we have to recall the important findings that short oligoribonucleotides possessing base sequences
complementary to that of the promotor sequence of the template
can specifically serve as primers in the initiation of RNA
13 14
chains ' . This, however, implies recognition between specific
sequences of primer and template by direct base-base interactions. Consequently we have to assume that a few nucleotides
downstream from the 3'-OH terminus of the growing RNA chain are
involved in the formation of a hybrid structure with the corresponding deoxyoligonucleotide sequence of the template. If the
primer site is occupied as proposed, the substrate site is
created. The selection of substrates is based on their ability
to fit into this hybrid structure. At first this involves physical interactions only, namely direct interactions between
substrate base and the base of the terminal nucleotide of the
3 -terminus of the RNA chain and interactions of the substrate
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with functional groups of the enzyme at the active site. After
bond formation and translocation of the enzyme, this nucleotide
becomes the new 3'-terminus of the hybrid structure. The least
stable situation occurs during the initiation of a new RNA chain,
when the initiating nucleoside triphosphate has to mimic the
3'-terminus of a growing RNA chain and together with the substrate forms the hybrid structure. This explains the highly unfavorable kinetic parameters measured in the synthesis of pppApU.
The structural elements forming the hybrid structure at the
transcriptional complex are: template, enzyme, part of the 3'end of the growing RNA chain and the bound substrate. The enzyme
provides at least the following functions within this complex
(1) specific recognition of the promotor sequence of a template,
(2) stabilization and proof-reading of the hybrid structure built
from template, primer and substrate at the enzyme active site and
(3) catalysis.
ACKNOWLEDGEMENT
Our thanks are due to our colleagues Drs. G. Rhodes, R. Clegg
and T.M. Jovln for stimulating discussions and helpful criticisms. We are indebted to Ms. S. Schr6der for skillful technical
assistance.
REFERENCES
1
2
3
4
5
6
7
8
9
Chamberlin, M.J. (1976) in RNA Polymerase, pp. 40-45, Cold
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Rhodes, G. and Chamberlin, M.J. (1975) J. Biol. Chem. 250,
9112-9120.
Mangel, F.W. and Chamberlin, M.J. (1974) J. Biol. Chem. 249,
2995-3001.
Johnston, D.E. and McClure, W.R. (1976) in RNA Polymerase,
pp. 413-428, Cold Spring Harbor Laboratory.
Zillig, W., Zechel, K. and Halbwachs, H. (1970) Hoppe Seyler's
Z. Physiol. Chem. 351, 221-224.
Sternbach, H., Engelhardt, R. and Lezius, A.G. (1975) Eur. J.
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1375-1380.
Krakow, J.S., Rhodes, G. and Jovin, T.M. (1976) in RNA Polymerase, p. 138. Cold Spring Harbor Laboratory.
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10 Segel, I.H. (1975) Enzyme Kinetics, pp. 646-650, Wiley-Interscience, New York.
11 Segel, I.H. (1975) Enzyme Kinetics, pp. 822-824, Wiley-Interscience, New York.
12 Rhodes, G. and Chamberlin, M.J. (1974) J. Biol. Chem. 249,
6675-6683.
13 Downey, K.B., Jurmark, B. and So, A. (1971) Biochemistry 9,
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14 Terao, T., Dahlberg, J. and Khorana, H.G. (1972) J. Biol.
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