Journal
of General Virology (1998), 79, 1033–1045. Printed in Great Britain
...................................................................................................................................................................................................................................................................................
Variation in ATP requirement during influenza virus
transcription
Klaus Klumpp, Martin J. Ford† and Rob W. H. Ruigrok
EMBL Grenoble Outstation, c/o ILL, BP 156, 38042 Grenoble Cedex 9, France
The ATP requirement of influenza A virus RNAdependent RNA polymerase was studied during
in vitro transcription reactions. In complete transcription reactions, the Km for ATP was 10-fold higher
than the Km values for the other NTPs. However,
during transcription elongation the Km for ATP was
as low as the Km values for the other NTPs,
suggesting a special requirement for ATP during
transcription initiation. Gel analysis of RNA products
of transcription initiation reactions showed that the
incorporation of AMP into nascent RNA was more
efficient at positions 4, 6 and 7 relative to the
template RNA than at position 5. The polymerase
produced short, abortive transcripts with lengths
corresponding to positions 3 and 4 relative to the
Introduction
RNA-dependent RNA polymerases (RdRps) are the key
enzymes for the multiplication of many RNA viruses. They
typically are relatively large, multifunctional proteins or
protein complexes responsible for virus specific, regulated
RNA synthesis during the transcription and replication of viral
genomic RNA. These enzymes catalyse reactions that are
essential for virus multiplication but they show considerable
differences from cellular RNA synthesizing proteins. They
therefore constitute potential targets for antiviral chemotherapy (Meanwell & Krystal, 1996 ; Tisdale et al., 1995). The
actual polymerase active site of RdRps may have evolved from
a common, ancestral polymerase module (Poch et al., 1990), but
Author for correspondence : Klaus Klumpp. Present address : Roche
Products Ltd, 40 Broadwater Road, Welwyn Garden City, Herts
AL7 3AY, UK.
Fax ­44 1707 332 053. e-mail klaus.klumpp!roche.com
† Present address : GlaxoWellcome Research and Development, Gene
Function Unit, Medicines Research Centre, Gunnels Wood Road,
Stevenage, UK.
template but never to position 5 or longer. These
results suggest that incorporation of AMP at
position 5 induces the influenza A virus polymerase
to go through a transition from a transcription
initiation to an elongation complex. This functional
change of the polymerase complex rather than a
requirement for ATP β–γ bond hydrolysis is the most
likely reason for the particularly high Km for ATP
during the early phase of transcription. This conclusion is supported by the fact that the ATP
analogue ATPγS [adenosine 5«-O-(3-thiotriphosphate)] can efficiently replace ATP in in vitro transcription reactions and shows a comparable drop of
Km between transcription initiation and elongation.
biochemically RdRps have not yet been as well characterized
as DNA-dependent polymerases or reverse transcriptases. A
better understanding of similarities and differences between
individual cellular and viral polymerases will support the
development of new antiviral strategies.
The influenza A virus genomic ribonucleoprotein (RNP)
constitutes the template for transcription by its specific RdRp.
The genome consists of eight different single-stranded RNA
segments (vRNAs) that all contain highly conserved and
partially complementary sequences of 13 nucleotides at the 5«
end and 12 nucleotides at the 3« end (Skehel & Hay, 1978 ;
Robertson, 1979 ; Desselberger et al., 1980 ; Stoeckle et al.,
1987). The genomic RNA is covered with nucleoprotein (NP),
which is responsible for the formation of the specifically coiled
ribonucleoprotein structure (RNP) with the RNA-bound NP
polymer folded back on itself in a looped structure (Compans
et al., 1972 ; Jennings et al., 1983 ; Ruigrok & Baudin, 1995). The
role of structural characteristics like the coiled RNP fold-back
structure (Compans et al., 1972 ; Jennings et al., 1983) and the
fact that NP binding to the genomic RNA exposes all
Watson–Crick positions of the bases to the solvent (Baudin et
al., 1994 ; Klumpp et al., 1997) for transcription activity is at
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K. Klumpp, M. J. Ford and R. W. H. Ruigrok
present still unclear. The influenza A virus specific RdRp is a
heterotrimeric protein complex composed of the subunits PA,
PB1 and PB2 (Lamb, 1989 ; Lamb & Krug, 1996). Transcription
activity can be followed in vitro with detergent disrupted virus
particles or purified RNPs in the presence of RNA primers
(Krug et al., 1989). In virions, the polymerase complex is bound
to the conserved 3« and 5« ends of the single-stranded genomic
RNA (Klumpp et al., 1997). Transcription activity is dependent
on the presence of these ends for transcription initiation (Krug
et al., 1989 ; Hagen et al., 1994 ; Fodor et al., 1994, 1995) and NP
binding to the template RNA is required for efficient elongation
(Honda et al., 1988).
In vivo, influenza virus transcription and replication occur in
the nucleus of infected cells (Herz et al., 1981 ; Jackson et al.,
1982). The RNA polymerase initiates transcription at the 3«
termini of the genomic RNA segments using as primer a 9–15
nucleotide capped RNA fragment derived from the 5« ends of
cellular mRNAs (Bouloy et al., 1978 ; Krug et al., 1979 ; Beaton
& Krug, 1981). It appears that the polymerase preferentially
uses RNA primers that are able to undergo base-pairing
interactions at least with the 3«-terminal uridine of the vRNA
template, although base-pairing between primer and template
is not essential for transcription initiation (Beaton & Krug,
1981 ; Krug et al., 1980 ; Lamb et al., 1981 ; Hagen et al., 1995).
The polymerase can also initiate transcription by using small
RNA oligonucleotides like ApG, GpG or ApGpC as primers,
which are complementary to the vRNA 3« end. There is no
obligatory site of transcription initiation. Depending on the
primer sequence, transcription initiation can occur with GTP or
CTP between positions 2–4 of the vRNA 3« end (Hagen et al.,
1994, 1995 ; Plotch et al., 1981 ; Honda et al., 1986).
We have analysed the nucleotide triphosphate requirements of the influenza virus RdRp during transcription
initiation and elongation. The Km for ATP in in vitro
transcription reactions is known to be at least 10-fold higher
than the Km values for the other three NTPs (Stridh & Datema,
1984), but the reason for this difference is unknown. Relatively
high Km values for specific purine nucleotides have been
documented before in studies with other RNA polymerases for
initiating NTPs (Testa & Banerjee, 1979 ; Anthony et al., 1969).
However, during primed transcription, the influenza virus
polymerase neither initiates with ATP nor is it dependent on
ATP for the formation of the first phosphodiester bond. We
have studied separate phases of influenza virus transcription in
vitro in order to better understand this specific requirement for
ATP.
Methods
+ Materials. Influenza virus A}PR}8}34 RNPs were prepared by
standard procedures as described (Klumpp et al., 1997). RNases for RNA
sequencing, SP6 RNA polymerase and unlabelled ribonucleoside 5«triphosphates were purchased from Pharmacia, S-adenosyl--methionine
(SAM), the dinucleotide ApG, kinase inhibitors and ATP analogues from
Sigma, radiolabelled ribonucleotide 5«-triphosphates (3000 Ci}mmol)
BADE
from Amersham and vaccinia virus guanylyltransferase from Gibco BRL.
Recombinant ribonuclease inhibitor RNasin was from Promega.
+ Assay of influenza virus specific transcription. Except when
indicated so in the figure legends, complete ApG transcription reactions
were performed at 30 °C in 50 µl reaction mixtures containing 0±1–0±4
nM RNP, 50 mM Tris–HCl, pH 7±8, 100 mM KCl, 10 mM NaCl, 10 mM
DTT, 0±4FS25 U}µl RNasin, 0±2 µg}µl BSA, 0±4 mM ApG, 0±5 mM GTP,
CTP, UTP and 1 mM ATP, keeping the α-$#P-labelled NTP at 50 µM for
the times indicated in the figure legends. Reactions were stopped by the
addition of 50 µl 25 % trichloroacetic acid (TCA), 2 % Casamino acids and
incubated for 30 min on ice. Samples were filtered through a Millipore
AP15 glass-fibre filter using a Bio-Rad BioDot microfiltration device and
washed with 10 % TCA. Retained radioactivity was measured by
scintillation counting.
Preinitiated polymerase transcription complexes were prepared by
incubating 0±4–0±8 nM RNP under the same conditions as described
above either in the absence of UTP or in the presence of 0±1 µM UTP for
1–10 min at 30 °C. The reactions were then passed through 1 ml
Sephadex G-50 columns (Pharmacia) equilibrated in transcription buffer,
leading to a 10&–10'-fold separation of NTPs and ApG from RNP.
Transcription elongation rates were measured after the addition of fresh
NTPs (0±5 mM) and [α-$#P]NTP at 50 µM. Reactions were stopped and
analysed as described above. For gel analysis of transcription products,
reactions were stopped by the addition of 40 mM EDTA. RNA was
purified by phenol extraction and ethanol precipitation following
standard protocols (Sambrook et al., 1989), resolubilized in denaturing gel
loading buffer (7 M urea, 20 % sucrose) and loaded onto 20 % acrylamide
gels containing 7 M urea. The polymerase elongation complexes were
strictly dependent on UTP for the resumption of transcription activity
and full elongation was dependent on the addition of all four NTPs,
indicative of an efficient separation of NTPs and primer during G-50
column chromatography. RNA primed transcription reactions were
performed under the same incubation conditions as described above
using 0±1 nM RNP, 60 nM capped GEM-RNA (see below) and NTP
concentrations as described in the figure legends.
+ In vitro RNA synthesis and RNA capping reaction. In vitro
transcription with SP6 RNA polymerase was done according to standard
procedures (Promega protocols and applications guide) using as template
DNA SmaI-digested pGEM-7Zf­ plasmid (Promega). The use of the
resulting 68 nucleotide GEM-RNA for influenza virus specific endonuclease reactions has been described previously (Hagen et al., 1994 ;
Chung et al., 1994). In vitro transcribed RNA was purified by
electrophoresis on 12 % acrylamide–7 M urea gels ; bands were visualized
by UV shadowing, excised and eluted overnight in 0±6 M ammonium
acetate, 1 mM EDTA, 0±1 % SDS. Unlabelled, capped RNA was produced
by performing the transcription reaction in the presence of 1±5 mM cap
analogue (m(GpppG, Boehringer), 0±3 mM GTP, which results in the
capping of " 50 % of the transcripts. Vaccinia virus guanylyltransferase
(Gibco BRL) was used to specifically label GEM-RNA in the cap structure.
RNA (10 µg) was incubated with 6 U guanylyltransferase in 50 mM
Tris–HCl, pH 8, 1±25 mM MgCl , 6 mM KCl, 5 mM DTT, 0±6 U}µl
#
RNasin, 1 mM SAM and 1 µM [α-$#P]GTP in a 30 µl volume for 1 h at
37 °C. The reaction was stopped with denaturing RNA gel loading buffer
and cap-labelled GEM-RNA was purified by denaturing gel electrophoresis as described above. Direct sequencing reactions of labelled RNA
with RNases T1 and U2 were done as described (Klumpp et al., 1997).
+ ATPase assays. For thin-layer chromatography, 1 nM purified
RNP was incubated with 2 nM [α-$#P]ATP (specific activity 3000
Ci}mmol) and 0±5 µM cold ATP in transcription buffer (see above) for
30–60 min at 30 °C in 20 µl total volume. Control reactions were
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ATP requirement of influenza virus transcription
performed with ATP in the absence of RNP (C-), with ATP in the
presence of 2 mU T4 polynucleotide kinase and 100 ng DNA oligonucleotide (C­, production of ADP) and with ATP in the presence of
limiting amounts of alkaline phosphatase (production of labelled ADP
and labelled phosphate, control for the absence of phosphatase activity in
RNP preparations). A 1 µl sample of the reactions was spotted onto
polyethyleneimine-coated TLC plates (Schleicher and Schuell) and
separation was in 1 M LiCl, 0±5 M formic acid. For HPLC analysis of ADP
production, complete transcription reactions, transcription initiation or
elongation reactions were performed as described above, but with all
NTPs at 0±5 mM concentration, RNP at 1 nM and using 100 µl reaction
volumes. Transcription reactions were stopped by the addition of EDTA
to 40 mM and loaded onto a 2 ml Waters Symmetry C18 column,
equilibrated with 0±1 M potassium phosphate, pH 6±5, 5 mM tetrabutylammonium hydroxide (TBA). Bound nucleotides were eluted with a
0–50 % methanol gradient in the same buffer.
Results
ATP requirements of the influenza virus specific
transcription reaction
We studied the kinetic parameters of influenza virus specific
RNA synthesis in a system using virion derived RNPs, which
represent preformed polymerase–template complexes, in the
presence of the dinucleotide primer ApG and various amounts
of nucleotide triphosphates (NTPs). Km values for the different
NTPs were determined under initial velocity conditions and
reproduced with independent virus preparations, RNP
preparations and separate batches of NTP stocks. Fig. 1 (a)
shows example plots of data fitted to a hyperbolic equation
using the least squares method or plotted in a linear fashion
according to the Hanes–Woolf equation (insets) (Cleland,
1979 ; Rudolph & Fromm, 1979). Km values were determined
from both hyperbolic and linear fits. The results are summarized
in Table 1. Lineweaver–Burk, Hanes–Woolf, Eadie–Hofstee
and direct plots gave comparable results in all experiments (the
deviations in calculated Km values were smaller than the
standard deviation between independent experiments). In
agreement with previous results using a different virus strain
(Stridh & Datema, 1984), we found that the Km for ATP in such
a complete transcription reaction was about 10-fold higher
than the Km values for the other three NTPs. Transcription
initiation occurs with CTP in this system (see template
sequence scheme in Fig. 4 c), but the Km for CTP was
comparable to the Km of GTP and UTP in contrast to studies
with other RNA polymerases that showed an increased Km for
the NTP used for the first phosphodiester bond formation,
either ATP or GTP (Testa & Banerjee, 1979 ; Anthony et al.,
1969 ; Osumi-Davis et al., 1992 ; Bonner et al., 1992).
The Km for ATP is reduced in elongation reactions
To determine if there was a specific requirement for ATP
during transcription initiation, we established a system to
perform initial velocity measurements of RNA synthesis
selectively during elongation by using preinitiated transcrip-
tion complexes separated from primer dinucleotides and NTPs
by size exclusion chromatography on G-50 columns (see
Methods). In vitro transcription reactions were performed with
purified elongation complexes in the presence of increasing
NTP concentrations and Km values were calculated by fitting
hyperbolic and linear equations to the data points as described
above (Table 1 and example curves in Fig. 1 b). We found that
in elongation reactions the Km for ATP was low and
comparable to the Km values for the other NTPs. When the Km
values for the NTPs in elongation reactions were compared to
complete transcription reactions only the Km for ATP had
significantly changed (16±9-fold decrease), whereas the Km
values for the other NTPs had decreased by a factor of only 3.
These results suggested that there was a specific requirement
for ATP during transcription initiation, which was different
from the requirement during elongation. There was a difference
in the maximal velocity of nucleotide incorporation between
complete transcription reactions and elongation reactions,
which was presumably caused by the loss of actively
transcribing polymerase complexes due to nonspecific adsorption to the column material (about 60 % of loaded protein
was recovered from the column) and to the dissociation of
stalled transcription complexes. The concentration of active
protein in the assay was therefore lower in elongation reactions
as compared to complete reactions.
A second ATP-dependent active site apart from the
polymerase active site could theoretically be involved in
transcription initiation, but not elongation. If this hypothetical
enzymatic function had a high Km for ATP, this could be a
reason for measuring a high Km (ATP) only in complete
transcription reactions, but not in elongation reactions.
Although influenza virus transcription initiation is independent
of ATP (see below and Figs 3 and 4), it would still be possible
that ATP was specifically required for promoter clearance.
Therefore, we performed a series of experiments to test if ATP
β–γ bond hydrolysis was required for influenza virus specific
transcription in vitro.
Influenza virus specific transcription in the presence of
β–γ blocked ATP analogues
ATP could be efficiently replaced by ATPγS [adenosine 5«O-(3-thiotriphosphate)] in influenza virus transcription
reactions, but no significant activity was detectable in complete
transcription reactions with either AMPPNP [5«-adenylyl(β-γimido)diphosphate] or AMPPCP (β-γ-methyleneadenosine 5«triphosphate) (Fig. 1 c). In elongation reactions, the apparent
Km values were low for both ATPγS and ATP, indicating that
they were used with equal efficiency by the polymerase (Table
1 and Fig. 1 c). Transcription activity, albeit about 5-fold lower
than with ATP, became detectable with AMPPNP, but not
with AMPPCP, in elongation reactions (Fig. 1 c). Similar
differences in the enzymatic activity have also been observed
with other polymerases in the presence of ATP analogues
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BADF
K. Klumpp, M. J. Ford and R. W. H. Ruigrok
(a)
(b)
Fig. 1. NTP requirements of the influenza
A virus RdRp for incorporation of α-32Plabelled CTP or GTP into TCA insoluble
material. (a) complete transcription, (b)
elongation, (c) ATP-analogues replacing
ATP in complete transcription (left) and
elongation (right). Transcription assays
were performed either with purified RNPs
or polymerase elongation complexes
obtained by preincubation of RNPs with
NTPs and primer and purified by G-50
size exclusion chromatography (see
Methods and legend to Fig. 2). RNP
(0±4 nM) was incubated with two
unlabelled NTPs at a concentration of
500 µM, 400 µM ApG, 50 µM of one
α-32P-labelled NTP and various
concentrations of the fourth NTP for
15 min at 30 °C. Km values were
estimated from fitting hyperbolic curves
to the data and linear regression analysis
respectively, and results are summarized
in Table 1. The insets show the same
data plotted according to the
Hanes–Woolf equation (s/v ¯ 1/
Vmax[s­Km/Vmax). The experiments
were reproduced with different virus and
RNP preparations using reaction times
between 5 min and 20 min. Incorporation
of labelled NTP was linear for at least 1 h
under these conditions.
(c)
(Testa & Banerjee, 1979 ; Fujioka et al., 1991 ; Lofquist et al.,
1993 ; Timmers, 1994). They are most likely due to a lower
affinity of the polymerase for imido and methylene analogues
of ATP than for ATP itself. These results could not formally
exclude the requirement for an ATP hydrolysing activity
during transcription initiation, although the efficient use of
BADG
ATPγS argued against this possibility. It was therefore
interesting to investigate if ADP was produced during
transcription initiation. We analysed the release of labelled
ADP from [α-$#P]ATP in transcription reactions by separating
the nucleotides on thin-layer chromatography sheets. We
found that, depending on the virus preparation, the cor-
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ATP requirement of influenza virus transcription
Table 1. Nucleotide requirements for influenza virus specific transcription in vitro
Km values for complete
reaction*
(initiation­elongation)
Km values for
elongation§
Reduction (-fold) in Km
after transcription
initiation
ATP
GTP
CTP
UTP
ATPγS
54³18† (9)‡
4±7³1±8 (3)
4±9³4±5 (6)
5±2³2±3 (4)
28³13 (5)
3±2³1±6 (8)
1±7³1±3 (3)
1±4³0±6 (3)
1±8³0±4 (2)
2±8³2±0 (3)
16±9
2±8
3±5
2±9
10
* ApG primed transcription reaction.
† The Km values in µM for the different NTPs as well as for the ATP analogue ATPγS, determined as described in the legends of Figs 1 and 3 for
ApG primed influenza A virus specific in vitro transcription reactions.
‡ No. of independent experiments from which the standard deviation was deduced.
§ Preinitiation of transcription with 400 µM ApG, 500 µM ATP, CTP, GTP, followed by size exclusion chromatography and incubation with
NTPs in the absence of primer.
responding RNP preparations could possess apparent ATPase
activity (Fig. 2 c). The ADP production, which varied considerably between virus batches, could also be analysed by
reverse-phase chromatography (Fig. 2 a, b). At the moment
it is not clear if this ATPase activity simply represents a
contamination of RNP preparations with a cellular protein or if
it has any significance for the virus. We could not correlate the
amount of copurifying ATPase activity with differences in the
transcription activity of RNP preparations. Nevertheless, to
exclude any interference with the present kinetic analysis, the
data presented in this paper were obtained from independent
RNP preparations, all without apparent ATPase activity (Fig.
2 a, c). These RNPs showed no apparent ADP production
during either transcription initiation or elongation suggesting
that ATP β–γ bond hydrolysis was not required for influenza
virus specific in vitro transcription (Fig. 2 a). Highly selective,
low level phosphorylation of a polymerase subunit may result
in ADP levels that could possibly escape detection in the
above control experiments. A kinase with characteristics of
casein kinase II has been found in variable concentrations in
virus particles (Tucker et al., 1990), but a possible function for
viral transcription was not investigated. We did not see any
stimulatory effect of adding casein kinase II to in vitro
transcription reactions, nor did DRB (5,6-dichloro-1-β-ribofuranosylbenzimidazole), an inhibitor of casein kinase II,
influence RNA synthesis by influenza virus RdRp at DRB
concentrations up to 200 µM (Table 2 ; Zandomeni et al., 1982).
AMP incorporation into RNA products occurs more
efficiently at position 4 than at position 5 of the vRNA
template
ATP usage during transcription initiation was studied in
more detail by analysing RNA products on denaturing
acrylamide gels. Transcription reactions were primed with
capped GEM-RNA (see experimental procedures). GEM-RNA
is cleaved by the influenza virus endonuclease to a so-called
G11 primer containing ApG at the 3« end (Hagen et al., 1994)
(Fig. 3 a, right panel, lanes ‘ RNP ’). The G11 primer is able to
base-pair to the first two bases of the vRNA template, and the
polymerase preferentially initiates transcription with CTP to
produce a 12 nucleotide RNA, G11­1 nt (Fig. 3 a, right panel,
lane ‘ RNP­CTP ’). This initiation reaction was then performed
with cap-labelled GEM-RNA and increasing concentrations of
CTP ; the reaction products were separated on acrylamide gels,
cut out and quantified by scintillation counting. The incorporation of the first nucleotide appeared to be very efficient
under these conditions and an apparent Km of 10 nM was
estimated from fitting a hyperbolic curve to the data points
(Fig. 3 a, left panel, and 3 b). Similar results were obtained using
unlabelled GEM-RNA and labelled CTP to study transcription
initiation. The initiated G11­1 nt RNA is shown again in Fig.
4 (left panel, lane 5). Corresponding reactions are shown on the
right panel of Fig. 4 after a longer exposure of the acrylamide
gel. The 12 nucleotide RNA could be further elongated with
either GTP (lane 4) or ATP (lane 2) to a 13 nucleotide RNA
(G11­2 nt). The RNA products migrated slightly differently
according to the nucleotides incorporated, which confirmed
that elongation was due to either AMP or GMP incorporation
at position 4 on the template RNA according to the sequence
heterogeneity of the 3« ends of the influenza A virus vRNA
segments at this position (see Fig. 3 c). Intermediate size
transcripts were produced in the presence of ATP, GTP and
CTP (Fig. 4, lane 1) and long transcripts in the presence of all
four NTPs (lane 3). Interestingly, AMP incorporation was
much more efficient at position 4 of the template (giving rise to
G11­2 nt) than at the subsequent template positions. In the
presence of CTP and ATP mainly 13 nucleotide RNA (G11­2
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BADH
K. Klumpp, M. J. Ford and R. W. H. Ruigrok
(a)
(b)
(c)
Fig. 2. Analysis of ATPase activity in RNP preparations. (a, b) Separation of nucleotides and small oligonucleotides produced in
RNP transcription reactions by reverse-phase HPLC. Peaks were identified by running nucleotide standards in parallel on the
same column. (a) Time-course of the synthesis of transcription initiation product ApGpC (AGC) in the absence (traces 4–7) or
presence (traces 8–11) of ATP. There was no detectable production of ADP, indicating the absence of ATPase or kinase
activity in the RNP preparation. (b) Reverse-phase HPLC separation of nucleotides and small oligonucleotides. Some RNP
preparations contained low level ATPase activity. For these preparations ADP was produced in the presence of RNP (traces 1,
3 and 5), but not in the absence of RNP (traces 2 and 4). (c) Thin-layer chromatography separation of nucleotides. Six
different RNP preparations originating from two different influenza A/PR/8/34 virus preparations were tested in in vitro ATPase
assays for the production of labelled ADP from [α32P] ATP. RNP preparations 1–3 showed no detectable ADP production,
whereas RNP preparations 4–6 from a different virus source produced low amounts of labelled ADP. ‘ C- ’ is a negative control
of [α3–2P]ATP incubated in ATPase reaction buffer, ‘ C­ ’ is a positive control showing [α-32P]ADP production from [α-32P]ATP
by 2 mU T4 polynucleotide kinase in the presence of 100 ng DNA oligonucleotide substrate. RNP preparations without
detectable ADP production [similar to the examples shown in (a) and (c), lanes 1–3] were used for kinetic analysis of in vitro
transcription.
nt) was produced, i.e. one CMP and one AMP incorporated,
although up to four AMPs could in theory be incorporated
into the nascent RNA according to the conserved vRNA
template sequence (Fig. 3 c). Also, much larger amounts of
short transcripts than long RNAs were produced in the
complete transcription reactions (lane 3). A large proportion of
the short RNAs represented abortive transcripts, because they
could not be elongated by the addition of excess NTPs. The
BADI
short transcripts were of 12 and 13 nucleotides length (G11­1
nt, ­2 nt), but, as indicated by the gap between these short
transcripts and intermediate size products (black line in Fig. 4),
transcription was much more processive as soon as the third
nucleotide had been incorporated, i.e. as soon as position 5 on
the template had been passed.
Incorporation of more than one AMP could be observed at
higher concentrations of ATP. We performed transcription
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ATP requirement of influenza virus transcription
Table 2. Transcription activity in the presence of DRB
ApG primed transcription reactions (50 µl total volume) were done as
described in experimental procedures, except that RNPs were
preincubated for 30 min after the addition of 1 µl of DRB (5,6dichloro-1-β--ribofuranosylbenzimidazole) dissolved in ethanol,
resulting in the indicated final concentrations of DRB. Transcription
reactions were started after the preincubation period by the addition
of NTPs and ApG.
DRB (µM)
Activity (%)
200
100
50
20
10
5
1
0
96
104
93
96
106
113
98
100
This is consistent with the observation that larger amounts of
short transcripts were produced as compared to longer RNA
products, and these shorter transcripts were stopped at position
4. Apparently, only a fraction of initiation events escaped into
the elongation phase by passing position 5 under these
conditions. These results suggest that a conformational change
of the polymerase is induced by the incorporation of the AMP
at position 5 relative to the template RNA. This incorporation,
which commits the polymerase to processive elongation,
requires higher concentrations of ATP than is required for
AMP incorporation at other template positions and explains
the measurement of a high apparent Km for ATP in complete
transcription reactions.
Discussion
initiation reactions under initial velocity conditions with
increasing amounts of ATP in the presence of GEM-RNA and
labelled CTP. As described before, products of 13 nucleotides
length (G11­2 nt) were detectable earlier than longer RNAs.
At higher ATP concentrations the immediate incorporation of
four AMP residues was observed (giving rise to G11­5 nt),
but no intermediate transcripts containing two or three AMP
residues incorporated were detectable (Fig. 5). At the highest
ATP concentration transcripts corresponding to G11­8 nt
were produced, presumably due to a misincorporation at
position 8 of the vRNA template (Fig. 5 and sequence scheme
in Fig. 3 c). These results suggest that the incorporation of the
third nucleotide into nascent RNA (position 5) is much less
efficient than incorporation of the second nucleotide (position
4), although ATP is used in both cases. The incorporation of
nucleotides beyond this critical position 5 of the template RNA
is again of higher efficiency as suggested by the absence of
intermediate products between position 4 and 7 of the template
(i.e. between products G11­2 nt and G11­5 nt). Similar
results were obtained with transcription reactions performed
with labelled GEM-RNA primer and unlabelled CTP (not
shown). Bands corresponding to G11­2 nt and G11­5 nt
were cut out of the gel and quantified by scintillation counting.
The relative band intensities plotted against the corresponding
ATP concentration could be fitted to a hyperbolic equation and
gave apparent Km values of 1 µM and 50 µM for the formation
of the G11­2 nt and the G11­5 nt products respectively
(Fig. 5). Interestingly, the apparent Km value for ATP measured
for the production of the G11­5 nt RNA was similar to the
‘ high ’ Km measured for ATP in complete ApG primed
transcription reactions (Table 1). These results indicate a
significant mechanistic difference between AMP incorporation
at position 4 as compared to AMP incorporation at position 5.
In the present study we established in vitro systems to
separately analyse and compare influenza virus specific transcription initiation and elongation reactions. The determination
of Km values for the NTP substrates of ApG primed RNA
synthesis revealed a specific requirement for ATP during
transcription initiation. In transcription elongation reactions
the Km for ATP was low and comparable to the Km values for
the other NTPs. The Km values for the NTPs other than ATP
were similar for both complete transcription and elongation
reactions. The significant change of the Km for only ATP in the
reactions suggested a specific requirement for ATP during
transcription initiation. The synthesis pattern of transcription
products as observed on denaturing acrylamide gels showed
that RNA synthesis by the influenza virus polymerase could be
connected to the production of significant amounts of short
abortive transcripts indicative of the transition between an
initiation and an elongation phase of transcription similar to
reports for other RNA polymerases (Carpousis & Gralla, 1980 ;
Hansen & McClure, 1980 ; Yamakawa et al., 1981 ; Luse &
Jacob, 1987 ; Levin et al., 1987 ; Martin et al., 1988 ; Sun et al.,
1996). The ability of the influenza virus polymerase to
reiteratively produce trinucleotide abortive transcripts in vitro
has been described before (Horisberger, 1982). The maximal
size of the influenza virus specific abortive oligonucleotides
corresponded to a stop of transcription at position 4 relative to
the template, i.e. the primer RNA plus two nucleotides in the
capped GEM-RNA system. The synthesis of much larger
amounts of transcripts, which are stopped at position 4,
compared to the amounts of longer RNAs was even more
intriguing when considering the fact that up to four AMP
residues could be incorporated at positions 4 to 7 relative to
the template (see Fig. 4), but one was incorporated more
efficiently than the others. An estimation of AMP incorporation kinetics at these positions under initial velocity
conditions showed that the apparent Km for ATP was low for
AMP incorporation at position 4, but high for the incorporation of further AMPs. The RNA synthesis reaction in the
experiment shown in Fig. 5 was limited by the omission of
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K. Klumpp, M. J. Ford and R. W. H. Ruigrok
(a)
(b)
(c)
Fig. 3. Endonuclease and transcription initiation reactions with cap-labelled GEM-RNA. (a) Left panel : transcription initiation
reaction performed with 0±1 nM RNP in the presence of increasing concentrations of CTP (0±0001, 0±0005, 0±001, 0±005,
0±01, 0±05, 0±1, 0±5, 1, 5, 10, 50, 100, 500 µM, from left to right) for 2 min at 30 °C. Right panel : transcription initiation
reaction performed with 0±1 nM RNP and 1 µM CTP (lane ‘ RNP,CTP ’), endonuclease reaction in the absence of CTP (lanes
‘ RNP ’) and RNA sequencing reactions of GEM-RNA using RNase T1 (G residues) and RNase U2 (A residues). Transcription
products were separated on a 20 % acrylamide gel. The G11 endonuclease product migrates slightly slower than the
corresponding RNase product in the T1 lane, because the former contains a hydroxyl group at the 3« end whereas the latter
has a phosphate group. The positions of the full-length GEM-RNA, the endonuclease product (G11) and the initiation product
(G11­1nt) are indicated on the left. Additional bands are due to unspecific degradation of the labelled substrate RNA under
the incubation conditions. (b) Band intensities of GEM-RNA, G11 and G11­1 nt were measured by cutting the corresponding
bands from the gel and scintillation counting. Relative band intensities of G11­1nt were plotted against CTP concentration and
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ATP requirement of influenza virus transcription
Fig. 4. Transcription initiation and complete transcription reactions with GEM-RNA and [α-32P]CTP. Short transcripts are
produced in large amounts and only until position 4 of the template (G11­1 nt, ­2 nt). RNPs (0±1 nM) were incubated with
60 nM capped GEM-RNA, 0±1 µM [α-32P]CTP and 10 µM NTPs for 15 min at 30 °C as indicated. Transcription products were
separated on 20 % denaturing acrylamide gels. The right panel shows an independent experiment under the same conditions
as the left panel, but with a longer exposure of the gel. Initiation products are indicated on the left ; the position of intermediate
size transcripts produced in the absence of UTP is marked with a black line on the left.
GTP and UTP from the reaction leading to a forced stop at
position 7 (G11­5 nt). The absence of RNA products
corresponding to positions 5 and 6 of the template therefore
suggests that passing position 5 is followed by a concomitant
increase of AMP incorporation efficiency. The apparent Km
(ATP) observed for passing position 5 is very similar to the
‘ high ’ Km (ATP) obtained in complete ApG primed transcription reactions, supporting the notion that the low
fitted to a hyperbolic curve. The graph shows the quantification of the first seven lanes (up to 0±1 µM CTP) to emphasize the
quality of the hyperbolic fit from low to saturating CTP concentrations. The apparent Km value for CTP for the first
phosphodiester bond formation was estimated to be 10 nM. The increase in band intensity for the formation of the G11­1
band at different CTP concentrations was linear for about 30 min under these conditions. (c) Nucleotide sequences of the G11
primer and the conserved influenza A virus vRNA 3« end, which is the immediate template for transcription. The G11 primer can
form two base-pairs with the template RNA, and transcription preferentially initiates with CTP. There is sequence heterogeneity
at position 4 of the template with segments 1–3, 5 and 7 containing C and segments 4, 6 and 8 containing U at this position
in the case of influenza virus A/PR/8/34. Transcription reactions were done in a mixture of all vRNA segments.
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K. Klumpp, M. J. Ford and R. W. H. Ruigrok
Fig. 5. Transcription initiation reactions
with GEM-RNA, ATP and [α-32P]CTP. Top :
0±1 nM RNPs were incubated with 60 nM
capped GEM-RNA, 0±1 µM [α-32P]CTP
and increasing amounts of ATP
(1–500 µM) for 5 min at 30 °C.
Transcription products were separated on
a 20 % acrylamide gel. Bottom : product
bands were cut out of gels, quantified by
scintillation counting and plotted against
the corresponding ATP concentration.
Apparent Km values for ATP for the
formation of two bands were estimated
by fitting a hyperbolic curve to the data.
efficiency step of AMP incorporation at position 5 accounted
for the ‘ high ’ Km for ATP measured in complete transcription
reactions. Interestingly, the influenza virus specific endonuclease activity, which provides primer RNA for transcription
initiation, was apparently not rate limiting in this RNA
synthesis system. The cleavage of precursor RNA and the
incorporation of the first nucleotide, CMP, was fast compared
to the production of a transcript having two nucleotides
incorporated.
We were able to detect this significant change in Km (ATP)
early in transcription, indicative of the transition of influenza
virus RdRp from transcription initiation to elongation, because
the specific sequence of the influenza virus vRNA 3« end
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requires AMP incorporation at four subsequent sites. The
present results pose the question whether this polymerase
transition also occurs at template position 5 with primers that
are initiated at position 2 instead of position 3, as is the case
with GEM-RNA. Such studies are in progress, and they will
determine if it is either the length of the nascent RNA passing
a threshold value or the incorporation of a crucial nucleotide at
a fixed position relative to the template that induces the
structural change of the polymerase.
Recent studies on eukaryotic RNA polymerase II suggest
that a β–γ hydrolysable ATP cofactor may be specifically
required for promoter escape during early transcription
(Goodrich & Tjian, 1994 ; Dvir et al., 1996). Evidence for a
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ATP requirement of influenza virus transcription
specific ATP requirement during transcription initiation was
also found in one study with E. coli RNA polymerase (Fujioka
et al., 1991), and was also described in association with the
requirement for specific initiation factors (Popham et al., 1989 ;
Kustu et al., 1991). Other RNA polymerases are independent of
ATP hydrolysis and even RNA polymerase II can function in
the absence of hydrolysable ATP analogues on supercoiled
DNA templates (Timmers, 1994 ; Goodrich & Tjian, 1994).
The requirement for ATP hydrolysis may therefore be
determined by the promoter type and template topology and
be due to the recruitment of accessory factors like nucleic acid
helicases or transcription enhancers rather than to a basic
function of nucleic acid polymerase active sites.
Is the transition from initiation to elongation, which we
observe with the influenza virus RdRp, coupled to ATP
hydrolysis ? Our present results show that transcription was
similarly efficient in the presence of either ATP or ATPγS,
arguing against the need for β–γ bond hydrolysis. ATPγS can
be used as a substrate by certain kinases, but the RNP
preparations showed no detectable kinase activity in control
experiments (see below). On the other hand, AMPPNP and
AMPPCP could not replace ATP in the same assay and the use
of AMPPNP, but not AMPPCP, for RNA synthesis became
measurable only in elongation reactions. The most likely
explanation for this result is an apparently low affinity of the
influenza virus polymerase for these ATP analogues, as has
been observed before in other polymerase systems (Testa &
Banerjee, 1979 ; Lofquist et al., 1993 ; Timmers, 1994 ;
Gershowitz et al., 1978). Transcription activity levels of
roughly 10 % with AMPPNP as compared with ATP would
remain undetected in our assays. The apparent increase in
affinity for AMPPNP during the elongation phase is consistent
with a conformational change of the polymerase, connected
with the change from a promoter-bound to a forward-moving
conformer.
Together, the findings describe an in vitro system to study
biochemical parameters of the influenza virus RdRp activity.
We observe a specific requirement for ATP during transcription
initiation and the production of distinct products of abortive
transcription indicative of an ATP-induced polymerase transition from transcription initiation to elongation at position 5
relative to the template RNA. This transition appeared to be
independent of ATP β–γ bond hydrolysis. It may be
mechanistically similar to the passage of pause sites by RNA
polymerases, which has also been shown to result in an
apparent increase in the Km value for the substrate needed to
induce polymerase translocation (Kingston et al., 1981 ;
Guajardo & Sousa, 1997).
identify a class of oligonucleotide inhibitors of the influenza virus RNA
polymerase. Proceedings of the National Academy of Sciences, USA 91,
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Cleland, W. W. (1979). Statistical analysis of enzyme kinetic data.
Methods in Enzymology 63, 103–138.
Compans, R. W., Content, J. & Duesberg, P. H. (1972). Structure of the
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We thank Dr Kjeld Larsen for help with the setting up of HPLC
protocols, Christine Vincent for ideas in chromatography and Drs
Isabelle Landrieu, Stefan Becker and Stephen Cusack for critical comments
on the manuscript. K. Klumpp was a recipient of an EMBL post-doctoral
fellowship.
Hagen, M., Chung, T. D., Butcher, J. A. & Krystal, M. (1994).
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