From the ¶ Department of Biological Sciences, State University
of New York, Buffalo, New York 14260 and the
Department of Chemistry, University of Massachusetts,
Amherst, Massachusetts 01003
Received for publication, August 16, 2002, and in revised form, October 23, 2002
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ABSTRACT |
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Unlike DNA polymerases, an RNA polymerase must
initiate transcription de novo, that is binding of the
initiating (+1) nucleoside triphosphate must be achieved without
benefit of the cooperative binding energetics of an associated primer.
Since a single Watson-Crick base pair is not stable in solution, RNA
polymerases might be expected to provide additional stabilizing
interactions to facilitate binding and positioning of the initiating
(priming) nucleoside triphosphate at position +1. Consistent with
base-specific stabilizing interactions, of the 17 T7 RNA polymerase
promoters in the phage genome, 15 begin with guanine. In this work, we
demonstrate that the purine N-7 is important in the utilization
of the initial substrate GTP. The fact that on a template encoding AG
as the first two bases in the transcript (as in the remaining two of the T7 genome) transcription starts predominantly (but not exclusively) at the G at position +2 additionally implicates the purine O-6 as an
important recognition element in the major groove. Finally, results
suggest that these interactions serve primarily to position the initiating base in the active site. It is proposed that T7 RNA
polymerase interacts directly with the Hoogsteen side of the initial
priming GTP (most likely via an interaction with an arginine side chain
in the protein) to provide the extra stability required at this unique
step in transcription.
The initiation of transcription imposes some unique mechanistic
requirements on an RNA polymerase. In contrast to events occurring during elongation, at the initial step of transcription initiation, two substrate nucleoside triphosphate molecules must
position accurately in the active site. Clearly a part of the binding
energetics is derived from Watson-Crick interactions between the
incoming bases and those in the template strand of DNA, but just as
clearly, base pairing interactions are not sufficient to provide the
binding energetics required for full function. Indeed, a single
Watson-Crick base pair is unstable in solution (1).
It is understood that for the Watson-Crick placement of the
elongating nucleotide (position +2 at initiation),
additional energy for binding of the nucleotide comes from interactions
between its triphosphate, magnesium, and protein functional groups
(2-5). This interaction would not be expected to be important in
binding the initiating (+1) nucleotide, and it has been shown in the T7 system that guanosine monophosphate and even the nucleoside guanosine have Km values comparable to or lower than that of
the triphosphate GTP (6). Some additional interaction(s) must be at play.
Most RNA polymerases show some preference for the initial base of the
transcript. Escherichia coli RNA polymerase promoters often
initiate with ATP, although some promoters begin with other NTPs at the
first position in the transcript (7, 8). Of the 17 phage RNA polymerase
promoters in the T7 genome, the canonical +1 position of 15 begins with
GTP, while two promoters begin with ATP (9). Recent studies have
demonstrated that T7 RNA polymerase initiates poorly on promoters
encoding A at position +1; transcription instead initiates
predominantly with an encoded G at position +2 (10-12). The SP6 enzyme
similarly demonstrates a 5-20-fold reduction in the level of
RNA production on promoters lacking an encoded G at position +1
(13).
The preference for initiation with GTP might suggest the importance of
a strong Watson-Crick base pairing interaction between the substrate
and the templating base (GC pairs being generally stronger than AT
pairs), but the lack of promoters that initiate with CTP might suggest
that this is not an important criterion. A remaining possibility is an
interaction of the protein with the incoming base itself. This would
explain the preference of a particular RNA polymerase for initiating
with specific bases. Indeed, a recent study has implicated
His-784 in contacting the 2-amino group of guanine in the minor
groove (12).
In the current work, we demonstrate that the preference for GTP as the
initiating nucleoside triphosphate by T7 RNA polymerase is indeed
likely the result of base-specific, non-Watson-Crick interactions. We
demonstrate that the N-7 and possibly O-6 positions along the major
groove of guanine are specifically involved in positioning the
substrate at position +1, explaining further the preference of this
enzyme to initiate with GTP and consistent with a preference for a
purine over a pyrimidine.
RNA Polymerase--
T7 RNA polymerase was prepared from E. coli strain BL21 carrying the overproducing plasmid pAR1219
(kindly supplied by F. W. Studier), which contains the T7 RNA
polymerase gene under inducible control of the lacUV5
promoter. The enzyme was purified, and its concentration was determined
( Oligonucleotides--
Oligonucleotides were synthesized by the
phosphoramidite method on an Applied Biosystems Expedite 8909 DNA
synthesizer. Single strands from a 1-µmol scale synthesis were
purified trityl-on using an Amberchrom CG-161cd reverse phase resin
(TosoHaas Inc.) as described previously (15). Purity of the
oligonucleotides was confirmed by denaturing (urea) gel electrophoresis
of 5'-end-labeled single strands. Double-stranded DNA was made by
heating complementary single strands to 90 °C, allowing the
resulting mixture to cool to room temperature over 2 h.
Kinetic Assays--
Steady-state assays of transcription were
carried out at 37 °C in a total volume of 20 µl. The resulting
mixture contained 30 mM HEPES (pH 7.8), 15 mM
magnesium acetate, 25 mM potassium glutamate, 0.25 mM EDTA, 0.05% (v/v) Tween 20 (Calbiochem, protein grade),
0.8 mM GTP or 7-deaza-GTP, 0.4 mM ATP, CTP, and
UTP each, and less than 0.06 µM
[ Product Assignment--
To properly assign all of the products,
at least three parallel reactions were performed for each promoter
construct. Conditions were as above, and reactions contained either
[ It has long been known that T7 RNA polymerase prefers to initiate
with guanosine as the first base in the RNA transcript (16-18). Recent
results have demonstrated a very strong preference for initiation with
GTP (C in the template strand, resulting in a 5' G) with a minor
ability to initiate with ATP (10, 12, 19).
It is well established that RNA polymerases, in general, face a
unique challenge at initiation, positioning the priming (+1) nucleotide
without the aid of stabilizing covalent and noncovalent interactions
with an upstream heteroduplex. Does the polymerase specifically bind or
position GTP as the incoming substrate in a manner unique to initiation?
The Guanine N-7 of the Initiating Nucleotide Is Critical--
The
most likely base-specific contacts with G within a GC pair lie
along the Hoogsteen face (20). To test the importance of a potential
Hoogsteen contact in the RNA polymerase substrate GTP, we have carried
out transcription in the presence of 7-deaza-GTP.
As illustrated in Fig. 1, this analog is
very similar to GTP with the exception that the nitrogen at the 7 position is replaced by a carbon (and the nitrogen lone pair is
replaced by hydrogen). As shown in Fig. 2, synthesis of a five-base
run-off transcript on a template encoding GGACU shows the 5-mer
as the predominant product using GTP as substrate. In contrast,
replacement of GTP by 7-deaza-GTP reduces synthesis of the run-off RNA
product on this template by more than 7-fold (Fig.
2, compare lanes 2 and 3).
The reduction in transcription in the presence of 7-deaza-GTP could
arise from a deficiency in the incorporation of this analog at either
the initiating or elongating positions during initiation (or on other
templates during subsequent elongation). To distinguish between these
possibilities, GMP was added to the reaction. It has previously been
shown that GMP can substitute for GTP at position +1, but of course,
since it lacks the triphosphate, it cannot substitute at any other
positions in the RNA (6). Lane 5 in Fig. 2 shows that the
inclusion of GMP in the mixture completely restores normal
transcription (note that due to the incorporation of a 5'-terminal
monophosphate, run-off transcripts initiating with GMP migrate more
slowly on the gel than normal products having a 5'-terminal
triphosphate group). This result demonstrates that 7-deaza-GTP
incorporates at position +2 as efficiently as GTP but is much less able
to incorporate at position +1. These results suggest a role for the N-7
nitrogen of guanine in positioning and/or binding of the initiating
NTP. In support of this conclusion, 7-methyl-GTP will also not
substitute for GTP in transcription initiation.1
The N-7 nitrogen of guanine does not appear to be important in
elongation synthesis, consistent with previous results that showed that
a more dramatic (cyanoborane) substitution at this position is
tolerated in dGTP as a substrate for various DNA polymerases (21) and
that 7-deaza-guanosine triphosphate can be used during preparative-scale enzymatic synthesis of RNAs more than 30 nucleotides long (22).
The Guanine O-6 Is Also Important for Initiation--
Conventional
wisdom has been that T7 RNA polymerase prefers to initiate with G but
can initiate well with A as the first base in the nascent transcript.
This would suggest that the purine O-6 is not important. This view
might be supported by the presence of two promoters in the T7 RNA
polymerase genome that encode A at position +1 (9). However, it has
recently been shown that on promoters that encode an initial sequence
AG ... , initiation begins primarily with G at position +2 (12,
19). To quantitatively probe this assertion, we have prepared an
otherwise consensus promoter construct that encodes the run-off
transcript AGGGA. As shown in Fig. 3,
lane 1, instead of the clearly defined five-base run-off
product dominating the longer products (as in lane 5), a
variety of RNA products are synthesized on this template. Comparison of
lanes 1-3 demonstrates that despite the encoding of A at
position +1, only a minor fraction of products initiate with ATP. The
molar distribution of RNA products formed in the presence of GTP and ATP is given in Table I and shows clearly
that only about 13% of all RNA products initiate with A; the remainder
misinitiate (at position +2) with G. This can be seen not only by
comparing the amounts of run-off products but also by comparison of the amounts of the various shorter abortive products.
Interestingly, as shown in lane 4 of Fig. 3 and compared
quantitatively in Fig. 4, in the presence
of GTP as the sole substrate, T7 RNA polymerase is capable of slippage
transcription (23) on the template that encodes AGGGA. Closer
examination of the results reveals significant differences between
slippage transcription on this alternate template and on the regular
template encoding GGGAA (Fig. 3, lane 8). In
particular, in slippage from the AGGGA promoter, there is a
consistently higher fraction of fall-off at each step in the slippage.
The increased fall-off at each step seen for the AGGGA-encoding
template could arise either 1) from a decrease in the rate of forward
synthesis at each step or 2) from an increase in the rate of complex
dissociation.
Examination of the data in lane 4 of Fig. 3 reveals that
there is less RNA produced overall on template 1 (compare with template 2, lane 8 in Fig. 3). This is consistent with the
first proposal that misinitiated G slippage leads to a significant
reduction in the rate at which the growing RNA chain carries out
forward slippage synthesis (in contrast, increased dissociation would lead to higher turnover and larger amounts of RNA).
Estimation of the Binding Strength of 7-Deaza-GTP--
To provide
better insight into the effect that 7-deaza-GTP has on the kinetics of
initiation, we attempted to measure the Km for
7-deaza-GTP at the initiating (+1) position by using a DNA promoter
construct encoding the run-off 4-mer GACU (6). Analysis of the results
presented in Fig. 5 turned out to be less
than straightforward. As expected, at very low concentrations of
7-deaza-GTP, misinitiation with ATP (at position +2)
predominates, but substantial misinitiation occurs even at high
concentrations of 7-deaza-GTP. In addition, extension of the
misinitiated products is less efficient as predicted above. As shown in
Fig. 5 (and in Fig. 6B), at
higher concentrations of 7-deaza-GTP, RNA synthesis (both short and
full length) actually decreases rather than leveling off as might be
expected. This makes the use of a simple binding equation
impossible.
In this context, correct initiation with 7-deaza-GTP competes directly
with misinitiation at position +2 (which produces pppApC). Therefore, if the K
This value can next be used to fit the data for production of
pppApC in Fig. 5 according to the following equation (24).
A critical function of an RNA polymerase is the selection of the
position of the transcription start site. From previous studies, it is
apparent that a significant part of the energetics stabilizing the
initially transcribing complex comes from protein interactions with the upstream duplex region of the promoter (25). Following the
separation of the DNA strands (melting), an RNA polymerase must
position the first two NTPs in the active site. It seems certain that productive positioning of the first two NTPs in the normally functioning initially transcribing complex occurs a minimal distance from the upstream duplex with this distance determined by the
distance along the protein between the tight binding region and the
enzyme phosphoryl transfer site (10). Initiation occurs primarily at
the 5th and 6th residues downstream of the duplex binding region (19)
(and with limited efficiency at the 4th position
(13)3).
The enzyme imposes a relatively strict minimal distance, but
not a sharp maximal distance, as very large non-nucleosidic
linkages still allow initiation at now very "distant" templating
bases (26). This accounts for the occurrence of downstream
misinitiation when the +1 templating base is a poor initiator either
because it does not encode G or because it does not have the optimal
environment (e.g. lacking a base 3' of
itself).4
Hoogsteen Functional Groups Are Important in Positioning the
Initiating Nucleotide--
Within the part of the DNA where initiation
could occur, the choice of initiation seems to be dependent on the
preference of T7 RNA polymerase for a particular initiating nucleotide.
It is apparent that this preference is not dominated simply by the strength of the Watson-Crick interactions with the complementary base
since mutation of the promoter to encode a C at this position leads to
a substantial reduction in activity (19). Indeed, only by using high
concentrations of CMP and keeping concentrations of all other
nucleotides well below the Km value for the
initiating nucleotide has it been possible to produce substantial amounts of transcripts initiating with C (27). A recent study has
implicated the 2-amino group of guanine in such stabilization (12). As
the current results show, GTP is preferred over ATP and dzGTP. The
latter result implicates the involvement of a major groove contact.
Replacement of nitrogen by CH at position 7 is not expected to
substantially reduce the strength of the Watson-Crick base pairing, and
although 7-deaza-guanine has been reported to have a lower dipole
moment than guanine (3.0 versus 8.0) it is oriented in the
same direction and seems unlikely to change the geometry of the
stacking of the initiating nucleotides (28). The most likely
explanation for the preference of the system for initiating with G, and
to a lesser extent with A, is the existence of protein contacts with
the Hoogsteen side of the initiating nucleotide. Correct positioning of
the nucleotide in the active site is likely to involve major groove
interactions with both the N-7 and O-6 of the guanine base. ATP and
dzGTP each retain only a part of this interaction (N-7 or O-6,
respectively) and so are disfavored compared with GTP.
Why Substrate Specificity?--
Since the stability of a single
base pair is likely insufficient to provide the energetics necessary
for correct positioning of substrate, it is easy to imagine how an RNA
polymerase would have evolved to provide additional stabilization for
binding and positioning of the initiating nucleotide. Note that DNA
polymerases, or RNA polymerases during elongation, do not need this
type of interaction because the equivalent "initiating" nucleotide
is covalently linked to the growing RNA or DNA primer and enjoys additional stabilization through base stacking with its covalently linked 5' neighbor. The problem is unique to de novo initiation.
These results suggest major groove interactions with both the N-7 and
O-6 groups of guanine. Among the amino acids, the most likely candidate
for simultaneous interaction with both the guanine N-7 and O-6
functional groups is arginine (see, for example, Ref. 29). The
available crystal structures of T7 RNA polymerase do not allow precise
prediction of the Arg in question; however, there are at least two
possible candidates: amino acids 425 and 632. These possess a
C
The effect of such stabilization is highlighted by the ability of T7
RNA polymerase to make dinucleotide RNA product on any long stretch of
DNA that has GG or GA encoded in the sequence (3'-CC-5' or 3'-CT-5' in
the template), although in the absence of the promoter these products
are not extended well (30,
31).5
These findings also allow us to speculate on the reason why most of the
phage T7 promoters start with the encoding of three G residues
at the beginning of the transcribed RNA sequence. In this case, the
dinucleotide product formed as the result of misinitiation at position
+2 is identical to the product of correct initiation. Since
positionally misinitiated products are elongated less efficiently, the
dinucleotide is released and can then be reused by priming at the
correct position (32).
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
280 = 1.4 × 105
M
1 cm
1) as described previously
(14). Purity of the enzyme was verified by SDS-PAGE.
-32P]GTP, [
-32P]ATP, or
[
-32P]ATP (PerkinElmer Life Sciences) as a label.
Reactions also contained 0.2 µM promoter DNA, 0.2 µM T7 RNA polymerase, and 0.4 mM GMP where
indicated. Reactions were incubated at 37 °C for 10 min and stopped
by addition of an equal volume of 95% formamide, 20 mM
EDTA (pH 7.8) gel loading buffer. The 3.0-µl aliquots were loaded
onto a 7 M urea, 18% polyacrylamide sequencing gel. After electrophoresis for 2.5 h at 2000 V/50 watts, gels were dried and
quantified using an Amersham Biosciences Storm 840 PhosphorImager. The percent fall-off was calculated for each band by
taking the ratio of the intensity (Ii) of the
band i corrected for the number of radioactive labels
incorporated (Ii/ni) and
dividing by the sum of corrected intensities of all the bands length
i and longer and multiplied by 100% (Equation 1).
(Eq. 1)
-32P]GTP, [
-32P]ATP, or
[
-32P] ATP (PerkinElmer Life Sciences) for labeling
purposes. For each band, the (molar) ratios of
[
-32P]GTP to [
-32P]ATP and of
[
-32P]ATP to [
-32P]ATP incorporation
were calculated. The bands were assigned on the basis of these ratios
and the approximate length of the RNA products. Results are shown in
Table I.
RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
View larger version (10K):
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Fig. 1.
Comparative structures of guanine,
7-deaza-guanine, and adenine.
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Fig. 2.
Run-off synthesis on a template encoding
GGACU. GTP, dzGTP, and/or GMP were present at 400 µM
as indicated. All reactions contained 400 µM each of ATP,
CTP, and UTP and 0.2 µM each of T7 RNA polymerase and
promoter DNA. Numbers within the gel represent the amount
(µM) of the transcript formed in a 15-min reaction at
37 °C. Assignment of the bands was carried out by comparing
mobilities and, in parallel experiments, the ratios of incorporation of
[ -32P]GTP and [
-32P]ATP for each band
as demonstrated more fully in Fig. 3.
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Fig. 3.
Transcription from the template encoding the
run-off transcript AGGGA compared with that from the control template
encoding GGGAA. Reactions contained 400 µM GTP and
400 µM ATP as indicated and 0.2 µM each of
T7 RNA polymerase and promoter DNA. Each reaction was run for 10 min at
37 °C as described under "Materials and Methods."
Identity and amounts of RNAs produced from the template encoding AGGGA
(template 1, from data in lanes 1-3 of Fig. 3)
View larger version (37K):
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Fig. 4.
Comparison of G slippage transcription
between two promoters from the data in lanes 4 and
8 of Fig. 3. Percent fall-off is
calculated as described under "Materials and Methods."
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Fig. 5.
7-Deaza-GTP concentration dependence of
transcription from a promoter construct encoding GACU. Reactions
contained 400 µM each of ATP, CTP, and GTP (with
[ -32P]ATP as label) and were run for 10 min at
37 °C.
View larger version (13K):
[in a new window]
Fig. 6.
A, determination of
Km for ATP in the synthesis of the dinucleotide
pppApC. B, 7-deaza-GTP concentration dependence of
transcription from the promoter construct encoding GACU showing
products pppApC (open circles) and all products
(filled diamonds). Each reaction was run for 10 min at
37 °C. The smooth line represents the best fit to the
competitive inhibition model (Equation 2) in which the
Ki of 750 ± 70 µM for
7-deaza-GTP represents its ability to competitively inhibit
misinitiation at position +2.
yielding a value for K
(Eq. 2)
The best fit of the data, shown in Fig. 6B, yields a
value of K
(Eq. 3)
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
that is less than 13 Å (the approximate length of an
extended Arg side chain) from the N-7 of the equivalent G (number 3) in
the crystal structure of the ternary complex (Protein Data Bank
code 1QLN).
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grant 1R01GM55002 and National Science Foundation Grant MCB-9630447.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Present address: The Scripps Research Inst., 10550 N. Torrey Pines Rd., La Jolla, CA 92037.
To whom correspondence should be addressed. Tel.:
413-545-3299; Fax: 413-545-4490; E-mail: CMartin@chem.umass.edu.
Published, JBC Papers in Press, November 9, 2002, DOI 10.1074/jbc.M208405200
1 E. Esposito and C. Martin, unpublished.
3 I. Kuzmine and C. Martin, unpublished.
4 I. Kuzmine, E. Esposito, and C. Martin, unpublished.
5 A. Újvári and C. Martin, unpublished.
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ABBREVIATIONS |
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The abbreviation used is: dz, 7-deaza.
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REFERENCES |
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