Reinitiated viral RNA-dependent RNA polymerase resumes replication at a reduced rate.

Abstract

RNA-dependent RNA polymerases (RdRP) form an important class of enzymes that is responsible for genome replication and transcription in RNA viruses and involved in the regulation of RNA interference in plants and fungi. The RdRP kinetics have been extensively studied, but pausing, an important regulatory mechanism for RNA polymerases that has also been implicated in RNA recombination, has not been considered. Here, we report that RdRP experience a dramatic, long-lived decrease in its elongation rate when it is reinitiated following stalling. The rate decrease has an intriguingly weak temperature dependence, is independent of both the nucleotide concentration during stalling and the length of the RNA transcribed prior to stalling; however it is sensitive to RNA structure. This allows us to delineate the potential factors underlying this irreversible conversion of the elongation complex to a less active mode.

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Published online 5 November 2008Nucleic Acids Research, 2008, Vol. 36, No. 227059–7067
doi:10.1093/nar/gkn836
Reinitiated viral RNA-dependent RNA polymerase
resumes replication at a reduced rate
Igor D. Vilfan1, Andrea Candelli1, Susanne Hage1, Antti P. Aalto2,
Minna M. Poranen2, Dennis H. Bamford2and Nynke H. Dekker1,*
1Kavli Institute of Nanoscience, Faculty of Applied Sciences, Delft University of Technology, Lorentzweg 1, 2628
CJ Delft, The Netherlands and2Institute of Biotechnology and Department of Biological and Environmental
Sciences, Viikki Biocenter, P.O. Box 56, 00014 University of Helsinki, Finland
Received August 27, 2008; Revised and Accepted October 14, 2008
ABSTRACT
RNA-dependent RNA polymerases (RdRP) form an
important class of enzymes that is responsible for
genome replication and transcription in RNA viruses
and involved in the regulation of RNA interference
in plants and fungi. The RdRP kinetics have been
extensively studied, but pausing, an important reg-
ulatory mechanism for RNA polymerases that has
also been implicated in RNA recombination, has
not been considered. Here, we report that RdRP
experience a dramatic, long-lived decrease in its
elongation rate when it is reinitiated following stal-
ling. The rate decrease has an intriguingly weak
temperature dependence, is independent of both
the nucleotide concentration during stalling and
the length of the RNA transcribed prior to stalling;
however it is sensitive to RNA structure. This allows
us to delineate the potential factors underlying this
irreversible conversion of the elongation complex to
a less active mode.
INTRODUCTION
Template-directed polymerization of nucleotides (NTPs)
is an essential process in all living entities. Accordingly,
enzymes catalyzing these processes operate in both cel-
lular organisms and in viruses. In RNA viruses, RNA-
dependent RNA polymerases (RdRPs) are the essential
catalytic components of the polymerization machinery.
RdRPs are also encoded by numerous cellular organisms,
where they initiate or amplify the regulatory mechanisms
known as RNA silencing (1). The structure and reaction
mechanisms of viral RdRPs display similarity with
many other nucleic acid polymerases, but nonetheless
incorporate subtle differences. For instance, viral RdRPs
adopt the ‘right hand-like’ conformation typical for
numerous nucleic acid polymerases, but they display a
distinct ‘closed-hand’ conformation, rather than the
more common ‘open-hand’ structure (2,3).
Viral RdRPs are capable of carrying out two distinct
reactions, replication and transcription. These reactions
are completed in four steps: (i) template recognition and
binding, (ii) initiation, (iii) elongation and (iv) termina-
tion. The binding of viral RdRPs to template RNA exhi-
bits characteristically low binding constants (4,5), but
binding may nonetheless be enhanced by specific nucleo-
tide sequences and/or RNA secondary structures (6,7).
During initiation and elongation, viral RdRPs perform a
nucleotidyl transfer reaction to polymerize the comple-
mentary RNA strand (8). While RNA or protein primers
may be required for initiation, most RdRPs initiate RNA
synthesis de novo (3).
To date, the kinetic studies of viral RdRP mechanism
have neglected the effect of viral RdRP stalling. Partial
RNA products isolated from poliovirus- and tobacco
mosaic virus-infected cells suggest that the RdRP indeed
frequently stalls, leading to compromised processivity
during RNA elongation in vivo (9). It has been suggested
that viral RdRP pausing could be brought about by spe-
cific RNA sequences and secondary structures (9), and is
likely a prerequisite for viral RNA recombination (10,11).
Furthermore, rational drug design against viral RdRPs
could benefit from the analysis of stalled viral RdRPs
(12). Finally, while stalling of viral RdRPs has been uti-
lized to separate the elongation and initiation stages of the
replication and transcription reactions (13), the conse-
quences of stalling on the elongation rates have not been
examined.
To quantitatively study the effect of stalling on the
kinetics of viral RdRPs, we have used the RdRP from
bacteriophage
?6(?6RdRP)
(Figure 1A). ?6 RdRP catalyzes primer-independent de
novo RNA synthesis on single-stranded RNA (ssRNA)
and double-stranded RNA (dsRNA) templates and
asa modelsystem
*To whom correspondence should be addressed. Tel: +31 15 278 3219; Fax: +31 15 278 1202; Email: n.h.dekker@tudelft.nl
Present address:
Andrea Candelli, Department of Physics and Astronomy, Vrije Universiteit, 1081 HV Amsterdam, The Netherlands
? 2008 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/
by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

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elongates with high processivity (14). During replication,
the single-stranded sense RNA strand [(+)RNA] serves as
the template for conversion to a dsRNA genome
(Figure 1B). In contrast, during transcription, the
(?)RNA strand within the dsRNA genome serves as the
template for a new (+)RNA strand, which leaves the ori-
ginal (+)RNA strand as a single-stranded by-product
(Figure 1C). In both processes, ?6 RdRP initiates exclu-
sively at the free 30-end of the template strand, which enters
the ?6 RdRP through the template tunnel leading from the
polymerase surface to the active site in the center of the
enzyme structure (Figure 1A). By contrast, internal initia-
tion of the elongation complex is not thought to occur
(Supplementary Figure S1) (15).
Here, we measure the rate of RNA elongation by ?6
RdRP and demonstrate that while it is possible to reiniti-
ate the polymerase following stalling induced by nucleo-
tide deprivation, the resulting elongation rate is drastically
and irreversibly reduced. This was quantified by measur-
ing the elongation rate of the reinitiated complex using
gel electrophoresis. Neither the NTP concentration nor
the length of the RNA synthesized prior to stalling had
an effect on the rate reduction following reinitiation.
We attribute the reduction in the elongation rate to a
transition to a sub-optimal conformational state of the
elongation complex, brought about by stalling, and
demonstrate that the conversion to the sub-optimal con-
formational state depends on the structure of the RNA
Figure 1. Bacteriophage ?6 RdRP performs replication and transcription. (A) A schematic of the ?6 RdRP structure. A nucleotidyl transfer active
site is linked to the enzyme surface via three tunnels: the template entry tunnel, the NTP tunnel, and the product exit tunnel. (B) During replication,
a complementary antisense RNA strand [(?)RNA; red line] is polymerized onto a sense RNA template [(+)RNA; black line]. The 30-end of the
template strand accesses the enzyme’s active site through the template tunnel. In the presence of NTPs, product dsRNA exits through the product
tunnel. (C) During transcription, the (+)RNA strand is displaced while ?6 RdRP polymerizes a new (+)RNA strand (green line) onto the (?)RNA
template. Here, the dsRNA genome is first unwound, and the (+)RNA strand is displaced at the entrance to the template tunnel, allowing only the
(?)RNA strand to enter the template tunnel. The dsRNA product exits through the product tunnel. The polarities of the RNA strands are indicated
in the schematics.
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template. In addition to the potential biological impor-
tance of the stalling, in vitro studies of polymerases have
used stalling to measure elongation rates (13,16–18). In
light of our findings, it is important to design the experi-
mental setup in a way that a possible reduction in the
elongation rate due to stalling is taken into account.
MATERIALS AND METHODS
Purification of therecombinant RdRP
from bacteriophage /6
The NdeI–EcoRI restriction fragment from pEM2 plasmid
(14) was transferred into a pET-28a(+) vector (Novagen,
USA). The resultant plasmid pAA5 was propagated in
Escherichia coli BL21(DE3) (19). ?6 RdRP was expressed
as previously described (14), except for 25mg/ml kanamy-
cin. The cells were harvested and resuspended in 50mM
Tris–HCl (pH 8.0), 300mM NaCl, 1mM imidazole and
disrupted. The supernatant was loadedonto a Ni-NTA affi-
nity column (Qiagen, Valencia, CA, USA). After two suc-
cessive washes with imidazole buffers (10mM and 20mM),
?6 RdRP was eluted in 50mM Tris–HCl (pH 8.0), 300mM
NaCl, 250mM imidazole. Further purification was done
using HiTrapTMHeparin HP and Q HP columns (GE
Healthcare, USA). ?6 RdRP was eluted with a linear
NaCl gradient (from 0.1M–1M NaCl) in 50mM Tris–
HCl (pH 8.0), 0.1mM EDTA. The purified protein was
stored in the elution buffer (containing 300mM NaCl)
at 48C.
Preparation of RNA molecules
ssRNAs were obtained via in vitro run-off transcription
using PCR-amplified sections of the pBB10 plasmid (20)
as described previously (primers listed in Table S1) (21).
To favor terminal incorporation of cytidine into the 30-end
of the transcripts used as templates for ?6 RdRP, the tran-
scription mixtures were supplemented with 20mM CTP.
dsRNA molecules were obtained by hybridizing com-
plementary ssRNAs in 0.5?SSC (Promega, Madison,
WI, USA) using a ‘gradual-cool’ temperature program
(21). dsRNA template for transcription reactions was
obtained by hybridizing three RNA molecules (4kb
RNA, 1.3kb RNA and 4kb ‘main’ RNA) in a molar
ratio of 1:1:1. Alternatively, a molar ratio of 1:4 was
used in hybridizations between longer and shorter comple-
mentary ssRNA molecules for calibration of the electro-
phoretic mobilities. All hybridized RNAs were purified as
described in (21).
A kineticstudy of elongation by reinitiation
of stalled /6RdRP
The elongation rate of ?6 RdRP was typically assayed in a
reaction mixture containing 80 nM RNA template, 2.6mM
?6 RdRP, 50mM HEPES pH 7.9, 20mM ammonium
acetate, 5mM MgCl2, 2mM MnCl2, 0.1mM EDTA
pH 8.0, 0.1% Triton X-100, 5% (v/v) Superase?In, and
2.5mM of each nucleotide. A 10-fold lower enzyme to
RNA template ratio was tested and shown to have no
effect on the kinetics of the reinitiated reaction (data not
shown). Prior to the reactions, RNA was heat-denatured
by incubation at 658C for 15min, followed by fast cooling
to 48C. The ‘stalled’ reactions were initiated using only
three NTPs (ATP, CTP, and GTP). The ‘unstalled’ reac-
tions were incubated in the absence of NTPs for the same
time period. After 15min incubation at temperature T1,
the temperatures of the stalled and unstalled reactions
were changed to temperature T2, and left to equilibrate
for 5min. The stalled ?6ECs were reinitiated by adding
UTP, and the unstalled reactions were initiated by the
addition of all four NTPs. Aliquots were taken at different
time points after the addition of missing nucleotides,
mixed with EDTA to 45mM final concentration, and
placed on ice. The products were analyzed using 0.75%
or 1.5% agarose gels for the transcription and replication
reactions, respectively. The agarose gels were preloaded at
2V/cm for 15min, and the electrophoresis was carried out
at 5V/cm at 48C. The reaction products were visualized
with ethidium bromide staining. The electrophoretic
mobilities of the RNA replication intermediates were
compared to a 2-log DNA ladder calibrated using a
seriesofRNAhybrids
Figure S2). For experiments including a heparin trap
(13), 9mg/ml heparin (Sigma-Aldrich, St. Louis, MO,
USA) was added after the stalling reactions, and the reac-
tions were incubated at 228C for 5min prior to the addi-
tion of UTP.
(SupplementaryDataand
RESULTS
Reinitiated /6ECreplicates withareduced rate
In replication, a ?6 RdRP elongation complex (?6EC) can
be stalled in vitro using a limited selection of NTPs and a
template molecule in which the 30-terminal region is devoid
of one or more of the nucleotides (Figure 2A; for proof of
stalling on short oligos, see Supplementary Data and
Figure S3). Elongation can then be reinitiated by the addi-
tion of the missing NTP(s), yielding an entirely double-
stranded product. To study the effect of stalling on the
rate of ?6 RdRP replication, we selected a 4193nt long
replication template (4kb ssRNA) in which the first occur-
rence of adenine was 50nt from the 30-end. The stalled
?6EC exhibited an electrophoretic mobility indistinguish-
able from that of free 4kb ssRNA (Supplementary
Figure S4). Following stalling and reinitiation by UTP
addition, aliquots were collected at successive time points
and analyzed on agarose gel (Figure 2B). After reinitiation,
a fraction of the replication template retained the electro-
phoretic mobility of free 4kb ssRNA (Supplementary
Figure 2B, lanes 2–10), which corresponds to either free
4kb ssRNA or to inactive stalled ?6EC. The electrophore-
tic mobility of successfully reinitiated ?6ECs decreased
with time as ?6 RdRP progressed along the 4kb ssRNA
(Figure 2B, lanes 2–10). Notably, the band corresponding
to the reinitiated ?6ECs stayed well-defined, suggesting
that the stalled ?6ECs reinitiated in a synchronized
manner. Termination of the replication reaction was
detected by a stabilization of the electrophoretic mobility
of the reinitiated ?6ECs (data not shown). This occurred
between 30min and 60min after reinitiation, from which
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we deduce an overall kelongbetween 1.2 and 2.3nt?s?1.
(For the discussion of the measured rates please see
Supplementary Data).
A control experiment was carried out to measure the
overall replication polymerization rate (kpoly) of unstalled
?6 RdRP under identical reaction conditions (Figure 2C).
To measure kpoly, the 4kb ssRNA was first incubated with
?6 RdRP for the same duration as above, but in the
absence of NTPs. Subsequently, all four NTPs were simul-
taneously added and aliquots were taken at successive
time points. At time zero, only free 4kb ssRNA could
be detected (Figure 2C, lane 1). In contrast to the reini-
tiated ?6ECs, at later times distinct bands could only be
detected for the free 4kb ssRNA and the final replication
product (4kb dsRNA) (Figure 2C, lanes 2–10), consistent
with an unsynchronized population of ?6ECs in this
experiment. The first appearance of 4kb dsRNA product
occurred 6min after the addition of NTPs (Figure 2C,
Lane 4), corresponding to a minimal kpolyof 12nts?1.
Surprisingly, the observed kpolyis at least six-fold higher
than kelong, despite the fact that kpolyis a composite of
kelongand the rates of accompanying initial stages of repli-
cation (e.g. ?6 RdRP binding and initiation of ?6EC).
This suggests that the randomly-initiated ?6EC elongates
considerably faster than the reinitiated ?6EC.
Anaccurate determination of kelongand kpoly
To support this initial observation, we obtained a more
quantitative determination of kelongand kpolyin the case of
reinitiated and randomly initiated ?6ECs, respectively. We
converted the electrophoretic mobilities of the elongation
intermediates of reinitiated ?6EC to a number of repli-
cated nucleotides by using a calibration curve relating
the two quantities (Supplementary Data and Figure S2).
We converted only the earlier time points in Figure 2B,
because the decreasing differences in the electrophoretic
mobilities in the later reaction stages precluded an accu-
rate determination of the number of replicated nucleo-
tides. The results show that the number of replicated
nucleotides after reinitiation increased linearly with time,
indicating that the reinitiated ?6EC exhibited a constant
kelong(Figure 3A, red points). kelongwas deduced from the
slope of the linear fit (Figure 3A, solid red line) and aver-
aged 2?1nts?1(mean and standard deviation deter-
mined from six experiments).
Independently, we determined an improved estimate for
kpolyfor randomly-initiated ?6RdRP replication, by extra-
polating the data to the minimum time ? necessary for the
conversion of a single 4kb ssRNA to its 4kb dsRNA
product, as previously reported (14). For example, to
extract the value of ? from the data in Figure 2D, the
normalized intensity of the band corresponding to
4kb dsRNA was plotted as a function of polymerization
time, and the experimental points were fitted to a straight
line (Figure 3B, solid red line). The x-intercept of the
linear fit yielded ? =287?42s, which corresponds to
kpoly=15?3nts?1
deduced from three experiments). These more accurate
values of kelongand kpolythus establish that the elongation
(meanandstandarddeviation
Figure 2. A reinitiated ?6 RdRP elongation complex (?6EC) and randomly-initiated ?6 RdRP replication show distinct electrophoretic profiles on
agarose gels. (A) Schematic of stalling and reinitiation of ?6EC. In the presence of three NTPs (ATP, GTP, CTP), a ?6EC is stalled at the 50th nt
from the 30-end of the template at temperature T1. The sequence elongated prior to stalling is shown in blue. After UTP addition, the stalled ?6EC
reinitiates and synthesizes the complementary strand at temperature T2. (B) Agarose gel of the elongation intermediates after reinitiation of the
stalled ?6EC on 4kb ssRNA template. Aliquots were taken at different times after reinitiation (telong). Letters S and P indicate 4kb ssRNA and 4kb
dsRNA, respectively. (C) Schematic of a randomly-initiated ?6 RdRP replication. 4kb ssRNA was incubated with ?6 RdRP, and all four NTPs
were subsequently added simultaneously. (D) Agarose gel of aliquots of randomly-initiated ?6 RdRP replication taken at different polymerization
times (tpoly).
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rate of randomly-initiated ?6EC exceeds that of reinitiated
?6EC by nearly an order of magnitude.
kelongof thereinitiated /6ECis independent oftemperature
To gain insight into the mechanisms underlying the
observed rate reduction, we compared the temperature
dependenceofkelong
andkpoly.To measure the
temperature dependence of kelong, we first stalled ?6EC
at temperature T1for 15min, equilibrated for 5min at
temperature T2, and reinitiated the ?6EC (Figure 2A).
We first fixed T1at 228C and varied T2(168C, 228C, or
308C) (Supplementary Figure S5). Using calibration
described above, we found that the number of replicated
nucleotides by the reinitiated ?6ECs increased linearly
with time for all three T2 (Figure 3A, green, red and
blue solid lines correspond to T2=168C, T2=228C,
and T2=308C, respectively). Surprisingly, we found
kelongto be independent of T2(kelongof 2?1nts?1for
all three T2). The experiment was then repeated at differ-
ent T1(168C and 308C) (Supplementary Figure S5). Here,
too, we found that kelongremained constant during repli-
cation and was unaffected by changes in T1. Furthermore,
the data reveals that kelongis independent of T1. In sum-
mary, we can conclude that over the range tested, kelongof
the reinitiated ?6EC is independent of temperature.
For comparison, we applied the same temperature var-
iation to the unstalled enzyme. As above, three different
values of T1and T2were tested (168C, 228C, and 308C for
both T1and T2) (Supplementary Figure S6). kpolywas
determined by measuring ? as in Figure 2D. At fixed
T1=228C, we found that kpolyincreased with increasing
T2, from 9?4nts?1at T2=168C, to 15?3nts?1at
T2=228C, and finally to 43?14nts?1at T2=308C
(mean and standard deviation deduced from three experi-
ments). No detectable changes in the measured kpolywere
observed when T1was changed to 168C or 308C (Supple-
mentary Figure S6). A comparison of kelongand kpolyis
revealing: first, kpoly is consistently greater than kelong
over the entire temperature range probed (Figure 3C):
the rate reduction is not a particularity of experiments per-
formed at room temperature; in addition, in contrast to the
temperature-insensitive kelong, thepolymerization rate kpoly
ofrandomly-initiated ?6ECshowedsignificantdependence
on T2(Figure 3C), from which we deduced an activation
free energy for the rate-determining step of 24 kBT by
fitting the data to an Arrhenius equation (solid line in
Figure 3C).
kelongisinsensitive tothe nucleotide concentration during
stallingand to thelength of elongatedRNA prior to stalling
To relate the observed differences between the kinetic pro-
files of reinitiated and randomly-initiated ?6ECs to speci-
fic regions of ?6RdRP structure, we investigated a number
of factors that could affect the interactions within the
?6EC. These experiments were performed at a fixed T1
and T2of 228C.
To determine whether the NTP concentration during
stalling affected the rate of the reinitiated ?6ECs, we
varied it from 0–2.5mM per NTP. The stalled ?6ECs
were reinitiated by the addition of a mixture of all four
NTPs to bring the final concentration of each NTP after
reinitiation to 2.5mM, as above. Aliquots were collected at
4minand 8minafter reinitiation andloaded onagarose gel
(Supplementary Figure S7A). In the complete absence of
NTPs during stalling, only the products of randomly-
initiated ?6ECs were detected, as expected (Figure 4A).
As the NTP concentration during the stalling stage was
Figure 3. Stalling of ?6EC reduces the elongation rate (kelong) following
reinitiation. (A) Progression of the reinitiated ?6EC along the 4kb
ssRNA at three different values of T2 (T2=168C, green triangles;
T2=228C, red circles; T2=308C, blue squares). Stalling of the ?6EC
was carried out at T1=228C. The number of replicated nucleotides (nt)
was determined from the electrophoretic mobilities of elongation inter-
mediates using the calibration curve in Figure S2C as described in the
Supplementary Data. Solid lines are linear fits to the data. kelongare
determined from the slopes of the corresponding linear fits. (B) The
relative concentration of the replication products as a function of the
polymerization time during randomly-initiated ?6 RdRP replication at
three different values of T2(T2=168C, green triangles; T2=228C, red
circles; T2=308C, blue squares). The incubation of 4kb ssRNA and ?6
RdRP prior to the addition of the NTPs was carried at T1=228C.
Values of ?, representing the minimal sum of the initiation and elonga-
tion times during randomly-initiated ?6 RdRP replication, were deter-
mined by the x-intercept extrapolated from the linear fits and used to
calculate the polymerization rates (kpoly) as described in the text. (C)
Arrhenius plot of the elongation and polymerization rates for the rein-
itiated ?6EC (open circles), and for randomly-initiated ?6 RdRP repli-
cation (filled squares), respectively. The data obtained with random
initiation was fitted to the Arrhenius equation (solid line).
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increased from 0.2mM to 0.6mM, this remained the case:
only the products of randomly-initiated replication were
present, and no detectable slow population of reinitiated
?6ECwasobserved(Figure4Atop,circles).However,once
the NTP concentration exceeded 0.8mM, the products of
randomly-initiated replication were no longer observed;
instead the slowly-migrating, distinct bands of elongation
intermediates attributable to reinitiated ?6ECs appeared
(Figure 4A top, triangles). Concentrations of reinitiated
?6ECs increased as the NTP concentration during stalling
was further increased to 1.4mM, but beyond 1.4mM no
further increase was detected. We note that little variation
was observed between the electrophoretic mobilities of the
reinitiated ?6EC, indicating similar kelong(Supplementary
Figure S7A).
Finally, to absolutely confirm that the bands detected
below 0.8mM NTP concentration during stalling were
indeed the products of randomly-initiated replication,
the experiments were repeated in the presence of heparin
following stalling (Supplementary Figure S7B). Heparin is
known to inactivate free (i.e. unstalled) RNA polymerase
(13), and can thus prevent random initiation after stalling.
We indeed observed the products of randomly-initiated
replication either entirely disappeared or were greatly
reduced in the presence of heparin (Figure 4A bottom,
circles). In contrast, the presence of heparin did not
reduce the concentration of the reinitiated ?6ECs; how-
ever, it decreased the nucleotide concentration during stal-
ling at which successfully reinitiated ?6ECs were detected
(Figure 4A bottom, triangles). We can thus conclude that
NTP concentration during the stalling stage affects the
likelihood of forming stalled ?6ECs, leaving the subse-
quent dynamics as captured by kelongentirely unaffected.
The site at which the ?6EC stalls on the template strand
determines the length of the dsRNA protruding from the
?6 RdRP product tunnel (Figure 1B). To investigate the
effect of the dsRNA length on the kelongof reinitiated
?6EC, we synthesized replication templates with a stalling
site at the third or seventh nucleotide. These replication
templates were designed to stall ?6EC in the presence of
three NTPs (i.e. ATP, CTP, and GTP) and reinitiate by the
addition of UTP, as in the case of 4kb ssRNA. On all these
templates, the number of replicated nucleotide increased
linearly with the elongation time (Figure 4B), as observed
previously. Similarly, the observed kelongwere measured to
be 2?1nts?1in all instances (mean and standard devia-
tion deduced from three experiments). Thus, we find the
dynamics of the reinitiated ?6EC to be insensitive to the
length of the elongated RNA prior stalling.
Kinetics ofthe reinitiated elongation complex during /6
RdRP transcription
Different secondary structures of replication and tran-
scription templates may interact differently with ?6
RdRP within the stalled ?6ECs (Figure 1), as RNA sec-
ondary structure elements located in viral RNAs have
been shown to regulate RdRP polymerization (22,23).
To test the role of template secondary structure on
?6EC kinetics after reinitiation, a 4kb long transcription
template was designed with a 50nt long single-stranded
sequence devoid of adenine at the 30-end of the transcribed
strand (Figure 5A). The design of the transcription tem-
plate included a nick solely to facilitate separation of the
template from the product on gel. After ?6EC stalling and
introduction of the missing UTP, ?6EC can reinitiate
yielding a branched elongation intermediates. At the end
of transcription, a completely double-stranded 3kb
dsRNA (Product A) and partially double-stranded RNA
(Product B) remained.
Similarly to the above study of ?6RdRP replication, we
measured kelong of the reinitiated ?6EC and kpoly of
Figure 4. Effects of the NTP concentration during the stalling stage and
the length of dsRNA synthesized prior stalling on kelongof the reinitiated
?6EC. (A) ?6 RdRP stalling was carried out at different NTP concentra-
tions, but the reinitiated ?6EC performed the reaction at a constant NTP
concentration of 2.5mM per NTP. The reinitiated ?6EC kinetics at each
NTP concentration were studied in the absence and presence of heparin.
Heparin was added after the stalling step, prior to reinitiation. Relative
concentrations of the replication products and intermediates at 8min
after the addition of UTP were obtained by dividing the intensity of the
corresponding band with the intensity of the 3kb dsDNA band of the
dsDNA ladder. In both plots, circles refer to the product of the randomly-
initiated replication (4kb dsRNA), whereas triangles correspond to the
replication intermediates of the reinitiated ?6EC (Supplementary
Figure S7). The experimental points were fitted to a sigmoidal curve.
(B) The number of nucleotide replicated prior stalling was 3, 7, or 50
nts and the data is indicated with circles, squares, and triangles, respec-
tively. Solid lines are linear fits to the data.
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randomly initiated ?6EC during transcription. First, kelong
on the transcription template after reinitiation was assayed
at three different T2(168C, 228C or 308C). The elongation
intermediates could be readily observed and displayed a
decreased electrophoretic mobility compared to the free
transcription template or the stalled ?6EC as a result of
their higher molecular weight and branched structure
(Supplementary Figure 5B). The electrophoretic mobility
of Product A corresponds well to that of 2876bp long
dsRNA, and the electrophoretic mobility of Product B
agrees with the value predicted by the previously construc-
ted calibration curves of RNA hybrids (Supplementary
Figure S2C). As in the replication reaction, an appreciable
fraction of the transcription template binds ?6RdRP, stalls
the ?6EC, and enables the synchronized reinitiation of the
stalled ?6EC after UTP addition, as judged from the well-
defined bands corresponding to the elongation intermedi-
ates (Figure 5B, lane 2). In these transcription experiments,
the more complex structure of the elongation intermediates
prevented determination of kelongfrom the electrophoretic
mobilities of the reaction intermediates. Rather, kelong
was determined by dividing the length of the transcribed
template by the typical time required to complete tran-
scription. This time was approximated by measuring the
mid-point between time t1, when the Product A band was
first detected, and time t2, when the intensity of the elonga-
tion intermediate was observed to decrease. We report
that kelongduring transcription was comparable to kelong
during replication at the lowest T2 (1.6?1.0nts?1at
T2=168C)butincreased
(4.6?1.5nts?1
T2=308C (red circles in Figure 5D) (mean and standard
deviation deduced from three experiments).
somewhatat higherT2
atat T2=228C) and 5.6?1.3nts?1
We then investigated the kinetics of transcription by
randomly-initiated ?6EC. In the transcription reaction
with the unstalled ?6EC, the transcription products
could readily be observed (Figure 5C). However, no
elongation intermediates were detected, due to a lack of
synchrony in the population of ?6ECs. We plotted the
intensity of the Product A band as a function of the
polymerization time and fitted the experimental points
to a line. The ? values obtained were used to determine
kpoly as above. At T1=228C, the kpoly obtained for
therandomly-initiated transcription
at T2=168C, kpoly=12?2nts?1at T2=228C, and
kpoly=11?2nts?1
Figure 5D). We thus observe that the values of kelongare
again consistently slower than the values of kpolyof the
randomly-initiated ?6 RdRP. However, the observed dif-
ferences are less pronounced than in the case of the repli-
cation reaction. In addition, in transcription, the kelong
and kpolydisplay similar temperature sensitivities, in con-
trast to the replication reaction, where considerable differ-
ences in the temperature trends were observed.
was5?2nts?1
at T2=308C (black squares in
DISCUSSION
We have established that a reinitiated ?6EC displays a
decreased kelongcompared to a randomly-initiated ?6EC.
The reduced kelong of the reinitiated ?6EC remained
constant during the replication of at least the first 2.5kb
of the 4kb ssRNA replication template (Figure 3A).
Furthermore, our estimate of the time point at which
replication of 4kb ssRNA was complete (Figure 2B)
yielded an overall elongation rate between 1.2nts?1and
Figure 5. Stalling affects the kelongduring transcription less than during replication. (A) Schematic representation of ?6 RdRP transcription with
stalling. The transcription template was synthesized by hybridizing three ssRNAs. The single-stranded sequence transcribed prior stalling is posi-
tioned at the 30-end of the template strand. ?6 RdRP is stalled with three NTPs at temperature T1, and transcription reinitiated by the addition of
UTP at temperature T2. When the reaction is complete, Products A and B result. (B) Agarose gel analysis of aliquots collected at different time
points after the reinitiation of the stalled ?6EC. The transcription template, Product A and Product B bands are indicated. Transcription inter-
mediates have the lowest electrophoretic mobility and can be observed at 20, 40, 60, 80and 100after reinitiation. (C) Transcription by ?6 RdRP in the
absence of stalling. Aliquots collected at different polymerization times were analyzed on agarose gel. (D) An Arrhenius plot of the experimentally-
observed elongation (red circles) and polymerization rates (black squares) of the reinitiated ?6EC and randomly initiated ?6 RdRP transcription,
respectively, during the transcription reaction.
Nucleic Acids Research, 2008, Vol. 36, No. 227065

Page 8

2.3nts?1, in a good agreement with the rate determined
for the replication of the first 2.5kb via fitting the elec-
trophoretic mobilities of the replication intermediate
(2?1nts?1). Thus, the reinitiated ?6EC elongates with a
reduced rate throughout the template, from which we con-
clude that the conversion undergone by ?6EC during the
stalling step is irreversible.
This irreversible conversion to the inhibited ?6EC state
must be promoted during stalling, as the initial stages
prior to ?6EC stalling proceeded similarly in both stalled
and unstalled reactions. An upper limit for the time
needed for the conversion to the inhibited ?6EC state
could be deduced from the shortest time required to
observe a synchronized population of reinitiated ?6ECs
(2min at T1=228C; Supplementary Figure S8). Analysis
of the temperature dependence of the reinitiated and ran-
domly-initiated ?6EC showed considerably different beha-
viour for the two complexes (Figure 3C). The kinetics of a
randomly-initiated ?6EC could be described well within
an Arrhenius model and yielded activation energy of 24
kBT for polymerization. In contrast, the kinetics of a rein-
itiated ?6EC showed little temperature dependence of the
elongation rates, implying that the conversion event
during the stalling step may be insensitive to temperature.
The irreversible conversion was similarly insensitive to
both the NTP concentration during stalling and the
length of the replicated template before stalling (Figure 4).
It is interesting to consider the possible nature of this
irreversible conversion. A comparison of replication and
transcription revealed that inhibition of the reinitiated
?6EC was appreciably greater with single- than double-
stranded templates (Figures 3 and 5). This suggests that
the template type has an effect on the conversion to the
inhibited ?6EC state. This may be akin to the regulation
of activity observed for other nucleic acid polymerases via
either specific (e.g. RNA hairpin-protein) or nonspecific
(e.g. electrostatic, hydrophobic) interactions (24–26). The
interactions between ?6 RdRP and its template that are
responsible for the conversion to the inhibited state do not
appear to be related to a particular RNA secondary struc-
ture, as the template strands were denatured prior to stal-
ling and were thus most likely present as an ensemble of
different RNA folds stable under the applied reaction con-
ditions. The fact that replication is affected more than
transcription may suggest the nature of the nonspecific
interactions involved (e.g. hydrophobic interactions pro-
moted by the exposed bases of ssRNA). However, the fact
that replication is affected more than transcription may
also implicate other nonspecific interactions that occur
with a higher probability in the case of ssRNA template
simply as a consequence of its lower persistence length,
which gives rise to a lower radius of gyration (27) and
thus a higher local concentration of the template at the
enzyme surface. Nonspecific RNA-?6 RdRP interactions
may cause the template RNA to interfere with the differ-
ent tunnels in the enzyme and impair their proper func-
tioning (e.g. they might limit exchange of free NTPs in the
NTP channel, exit of the dsRNA in the product tunnel,
or entry of the template strand in the template tunnel),
resulting in a decrease in the overall elongation rates.
Alternatively, RNA-?6 RdRP interactions could trigger
conformational changes within the ?6RdRP that result
in suboptimal reaction rates.
It is evident that complex enzymes such as ?6 RdRPs
are regulated through multiple mechanisms, and their sum
total dictates both the enzyme rates. It is thus intriguing
that even a simple stalling event can lead to considerable
changes in enzyme dynamics. It remains to be seen
whether the reduction in elongation rate after stalling is
a general property of viral RdRPs. The arrest of the elon-
gation complex could be biologically relevant during the
viral life cycle, in which RNA replication and transcrip-
tion are highly regulated (28). In addition, it could affect
the frequency of RNA recombination and thus the adap-
tation of RNA viruses to their environment (10,11). More
technically, many experiments have applied stalling
protocols to measure elongation rates of polymerases
(13,16–18). As shown here for ?6 RdRP, these measure-
ments may provide inaccurate values, in the absence of
confirmation that the enzyme is unaffected by stalling.
For example, when T7 RNA polymerase (T7 RNAP)
was stalled and subsequently reinitiated, the resulting
elongation rate was as low as 2nts?1as judged from a
study by Ferrari and co-workers (17). By contrast,
unstalled T7 RNAP exhibited elongation rates that range
from 40–400nts?1(29–31). Finally, polymerases may not
be the only enzymes whose activity can be regulated via
stalling. For instance, Kowalczykowski and co-workers
recently showed that RecBCD helicase switches lead
motors in response to the stalling at a specific DNA
sequence, similarly resulting in a rate reduction of motor
translocation (32).
SUPPLEMENTARY DATA
Supplementary Data are available at NAR Online.
ACKNOWLEDGEMENTS
We thank W. Kamping and R. Tarkiainen for their experi-
mental work and R. Tuma for fruitful discussions.
FUNDING
Nanoned, The Netherlands Organization for Scientific
Research, and the European Science Foundation (grants
to N.H.D.); the Finish Center of Excellence Program
2006-2011 (1213467 to D.H.B.), the Academy of Finland
andNanotechnology(700036
Graduate SchoolofBiotechnology
Biology (funding of A.P.A.). Funding for open access
charge: The Netherlands Organization for Scientific
Research.
to D.H.B.);
and
Helsinki
Molecular
Conflict of interest statement. None declared.
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