Crystal structure of RNA helicase domain from
genotype 1b hepatitis C virus has been determined at 2.3 Å resolution
by the multiple isomorphous replacement method. The structure consists of three domains that form a Y-shaped molecule. One is a NTPase domain
containing two highly conserved NTP binding motifs. Another is an RNA
binding domain containing a conserved RNA binding motif. The third is a
helical domain that contains no
-strand. The RNA binding domain of
the molecule is distinctively separated from the other two domains
forming an interdomain cleft into which single stranded RNA can be
modeled. A channel is found between a pair of symmetry-related
molecules which exhibit the most extensive crystal packing
interactions. A stretch of single stranded RNA can be modeled with
electrostatic complementarity into the interdomain cleft
and continuously through the channel. These observations suggest
that some form of this dimer is likely to be the functional form that
unwinds double stranded RNA processively by passing one strand of RNA
through the channel and passing the other strand outside of the dimer.
A "descending molecular see-saw" model is proposed that is
consistent with directionality of unwinding and other physicochemical
properties of RNA helicases.
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INTRODUCTION |
Helicases are enzymes catalyzing strand separation of double
stranded DNA (dsDNA)1 or
dsRNA coupled with hydrolysis of NTP. They are required for many
cellular events including transcription, RNA processing, translation,
and DNA or RNA replication (1-3). Since the first discovery of DNA
helicase activity more than 20 years ago (4, 5), many different
helicases have been identified with preferences for unwinding duplexes
of DNA or RNA. In in vitro experiments, nearly all helicases
require a single stranded region; some require a 3' overhang region (3'
to 5' helicases), whereas others require a 5' overhang region (5' to 3'
helicases). This single stranded region is proposed to provide an
initiation site for unwinding duplex nucleic acids (6).
Escherichia coli Rep DNA helicase, an extensively studied
helicase, exhibited highly processive unwinding of replicative form of
phage DNA in an in vitro experiment (7). In order for a
helicase to unwind duplex nucleic acids in a processive manner, the
enzyme should destabilize the hydrogen bonds between the base pairs,
translocate to the next base paired region, and repeat the cycle
without fully dissociating (6, 8). Recently, oligomeric forms,
generally dimers or hexamers, were observed for some DNA helicases (8,
9). These oligomers are believed to provide the helicases with multiple
nucleic acid binding sites necessary for the helicase function (6).
Although Rep DNA helicase, for example, is a stable monomer in solution
in the absence of DNA, a dimeric form of Rep is induced in the presence
of DNA which is known as the functional form (6, 8).
Hepatitis C viruses (HCVs) are the major etiologic agents of non-A,
non-B hepatitis that are estimated to have infected about 1% of the
population worldwide. HCV belongs to flaviviridae, the positive-strand
RNA virus family (10). Its genome consists of about 9400 nucleotides
with the gene order of N'-C-E1-E2-NS2-NS3-NS4A-NS4B-NS5A-NS5B-C' (10)
encoding a viral polyprotein of about 3010 residues (11). The
polyprotein is processed into functional proteins by host- and
virus-encoded proteases. Among the processed proteins, NS3 is best
characterized. The N-terminal one-third of NS3 is a serine protease
domain (12) which is known to cleave the NS3/4A, NS4A/4B, NS4B/5A, and
NS5A/5B junctions (13-15). The C-terminal two-thirds of NS3 is an RNA
helicase domain exhibiting nucleotide triphosphatase/RNA helicase
activity (16, 17). The domain was shown to unwind not only dsRNA but
also RNA/DNA heteroduplex and dsDNA (18). For its function the helicase
domain strictly requires a 3' overhang region, and it unwinds double
stranded nucleic acids only in the 3' to 5' direction (18).
Sequence alignment of many RNA helicases revealed four highly conserved
sequence motifs, and in HCV RNA helicase they are conserved as
G207SGKST, D290ECH, T322AT, and
Q460RRGRTGRGRRG sequences. The G207SGKST
sequence, known as Walker A motif, is found in nearly all NTP
hydrolyzing enzymes and is responsible for NTP binding. The D290ECH sequence is a variant of Walker B motif (19).
Biochemical and mutational analyses showed that the T322AT
sequence is important in unwinding of RNA, whereas the
Q460RRGRTGRGRRG sequence is important in the RNA
binding and the unwinding of RNA (20, 21).
Here we report the crystal structure of the genotype 1b HCV RNA
helicase domain and discuss in detail the structural features of the
conserved motifs. Based on a modeling experiment we propose a mechanism
of processive unwinding of the duplex RNA consistent with previously
observed physicochemical properties of the enzyme.
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MATERIALS AND METHODS |
Protein Purification and Crystallization--
The HCV RNA
helicase domain was isolated from an overexpressing E. coli
strain (BL21 (DE3)) and purified using a Ni-NTA-agarose column (QIAGEN)
and a poly(U)-Sepharose column (Amersham Pharmacia Biotech)
successively as described previously (22). Crystals were grown at
4 °C from a precipitant solution containing 30% polyethylene glycol
4000, 0.1 M sodium cacodylate (pH 6.5), and 0.2 M ammonium acetate on micro batch plates under Al's oil
(Hampton Research). The crystals belong to the space group
P3121 with the unit cell dimensions of a =
b = 93.3 Å, c = 104.6 Å. The crystals contain one molecule of the enzyme in the asymmetric unit.
Data Collection, Structure Determination, and
Refinement--
All diffraction data were measured from flash-frozen
crystals on a DIP2020 area detector system with graphite monochromated CuK
x-ray generated by a MacScience M18XHF rotating anode generator operated at 90 mA and 50 kV. Data reduction, merging, and scaling were
accomplished with the programs DENZO and SCALEPACK (23). Initial
diffraction phases were obtained by multiple isomorphous replacement
with three heavy atom derivatives. A difference Patterson map of an
iridium derivative (K3IrCl6) and that of
thimerosal derivative were calculated, respectively, with the fast
Fourier transform of the CCP4 suite (1994). Heavy atom sites for the
two derivatives were readily identified by strong Patterson peaks (>6
) on Harker sections (Z = 1/3) and at general positions.
The heavy atom positions were used to calculate MIR phases with the program MLPHARE (24). The MIR phases revealed relatively weak heavy
atom positions for a gold derivative (KAu(CN)2). The MIR phases with all three derivative data had a mean figure of merit of
0.55 at 3.0 Å resolution (Table I) and
were improved with real space density modification using the program DM
in the CCP4 suite (1994). The final MIR map was of high quality showing
virtually all side chain electron densities except in flexible regions. A nearly complete model of the helicase was built using the program O
(25) and was refined using X-PLOR program package (26). MIR phases were
abandoned at this point, and electron density maps calculated with
phases derived from the refined model allowed model building of loop
regions. The N-terminal 39 residues including hexahistidine tag
attached to the protein exhibited no electron density and were omitted
in the final model. Only faint electron densities were observed for
residues 416-420.
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Table I
Crystal structure determination and refinement statistics
Rsym = |Iobs Iavg|/ Iobs.
Rscale = FPH| |FP / |FP|. Phasing
power = r.m.s. (|FH|/E), where
E is the residual lack of closure error.
Rcullis = |E|/
FPH| |FP . Figure of
merit = < P( ) ei / P
( )>, where is the phase and P( ) is the phase
probability distribution. The Rfree was calculated
with 5% of the data.
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RESULTS AND DISCUSSION |
Protein Fold and Structural Features--
The structure of HCV RNA
helicase consists of three nearly equal-sized domains that form a
Y-shaped molecule (Fig. 1). The N-terminal one-third of the enzyme is a NTPase domain consisting of a
typical central core of pleated sheet surrounded by helices (27). The
active site cleft for the NTP hydrolysis can be readily identified at
the periphery of the domain by the G207SGKST sequence, the
NTP binding motif. The second domain is an RNA binding domain
containing the highly conserved Q460RRGRTGRGRRG sequence
identified as an RNA binding motif (Fig. 1). The folding pattern of the
RNA binding domain is similar to that of the NTPase domain, but it
contains fewer
-helices. The C-terminal one-third of the enzyme is a
helical domain composed of five
-helices and loops. The NTPase and
the helical domain are more or less continuously linked with a shallow
groove between the two, but the RNA binding domain is distinctly
separated from the other two domains forming a deep interdomain cleft
as shown in Fig. 1. The size of the interdomain cleft is adequate for
binding ssRNA (or ssDNA) but too narrow for binding double stranded
nucleic acids. The NTPase and the RNA binding domain are connected by two random coils (Fig. 1). In contrast, two antiparallel
-strands, unusually protruding from the RNA binding domain, are inserted into the
helical domain like an anchor linking the two domains (Fig. 1). The
turn region of the antiparallel
-strands is rich in hydrophobic
amino acids and interact extensively with apolar residues of the
helical domain.

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Fig. 1.
Ribbon drawing of HCV RNA helicase. The
NTP binding site is a cavity shaped by loops and -strands which are
discontinuous on the amino acid sequence. The TAT sequence is a part of
a loop connecting the NTPase and the RNA binding domain. The RNA
binding motif comprises the end of an -helix and the following loop.
Both the NTPase domain and the RNA binding domain face the interdomain
cleft.
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It remains a question why NS3 containing two completely different
activities, protease and helicase activities, is not cleaved into two
polypeptides in the processing of the viral polyprotein. In the crystal
structure presented here, the electron density for the first 39 amino
acid is not visible, indicating that the protease domain and the
helicase domain of NS3 should be linked to each other by a highly
flexible loop region. The visible N terminus of the helicase is at the
back side of the molecule (opposite the interdomain cleft), and thus
the enzyme activities of the protease and the helicase domain appear
independent of each other (see below).
NTP Binding Site--
The NTP binding site is located at the
periphery of the NTPase domain. The G207SGKST and the
D290ECH sequences are close to each other, lining part of
the active site cavity (Fig. 2). The side
chain of Asp290 is involved in an ionic interaction with
Lys210 and a hydrogen bond with the side chain of
Ser211. The D290ECH sequence is on an unusual
loop structure which orients the side chains of Asp290,
Glu291, and His293 toward the cavity and that
of Cys292 in the opposite direction (Fig.
3). The functional roles of
Asp290 and Glu291 can be inferred from the
structures of other NTPases. The crystal structures of Bacillus
stearothermophilus PcrA (28) and E. coli Rep DNA
helicases (29) were determined as complexes with ADP. It has been
proposed that in the major domain of RecA, which exhibits similarity to
the NTPase domains of the DNA helicases, the magnesium ion of Mg-ATP is
coordinated by Asp144 (30). This residue corresponds to
Asp223 and Asp214 in the DEXX motif
of PcrA and Rep DNA helicase, respectively. Glu215 (in Rep)
and Glu224 (in PcrA) of the DEXX motif are in
the same relative position in space as Glu96 in RecA, which
was proposed to activate the catalytic water molecule during the
hydrolysis of ATP. When the NTPase domains of HCV RNA helicase and PcrA
DNA helicase were superposed, Asp290 and Glu291
of HCV RNA helicase occupy relatively the same space as do
Asp223 and Glu224 of the DNA helicase (data not
shown), although overall structures of the two enzymes are quite
different. This observation implies that the two residues probably
share the same functions in the hydrolysis of NTP with the
corresponding residues in the DNA helicases and RecA. Consistently,
studies of the related DEXH helicase from vaccinia virus
demonstrated that substitution of Asp296 or
Glu297 in the DEXH motif with alanine abolishes
the NTPase activity and the helicase activity without affecting the RNA
binding affinity (31). The electron density for the NTP binding site is
very strong showing detailed features including many bound water
molecules (Fig. 2). However, it was not possible to predict a correct
binding mode of NTP by simple model building due to severe steric
clashes. Some conformational change in the active site cavity is
expected to occur upon NTP binding.

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Fig. 2.
Stereoview of the NTP binding site of HCV RNA
helicase. Three amino acids in the D290ECH sequence
are labeled. Cys292, pointing at the back of the figure, is
not visible. The bound water molecules are shown in red
spheres. The 2Fo-Fc electron density map was
calculated with the final refined coordinates at 2.3 Å and contoured
at 1.2 .
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Fig. 3.
Interaction of the D290ECH and
the T322AT sequence of HCV RNA helicase. Note
Asp290, Glu291, and His293 that
form a part of the active site cavity are on a loop structure and point
in the same direction. The backbones of the DECH motif and TAT sequence
are in cyan and magenta, respectively, and the
side chains are in green. The white dotted line
indicates the hydrogen bond between His293 and
Thr322.
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Flexible Hinge--
One of the two loops connecting the NTPase and
the RNA binding domain contains the invariant T322AT. In
the structure presented here, the hydroxyl group of Thr322,
located at the beginning of the loop (Fig. 1, 3), is involved in a
hydrogen bond with the imidazole ring nitrogen of His293.
In contrast, the hydroxyl group of Thr324, located toward
the middle of the loop and about 4 Å apart from His292, is
just exposed to the bulk solvent (Fig. 3). It is generally believed
based on mutational studies that the T322AT sequence
couples the NTP hydrolysis and the duplex unwinding by the enzyme (20).
In other experiments, H293A mutation in HCV RNA helicase and the
corresponding mutation in vaccinia virus RNA helicase were
shown to affect severely the duplex unwinding activity without
affecting the NTP hydrolysis activity (31, 32). Thus,
His293, Thr322, and Thr324 may
function as a triad in coupling the NTP hydrolysis and the helicase
activity. It is possible that His293 could switch its
hydrogen bond to and from Thr322 and Thr324
during the helicase function, which should require a small flexible hinge motion of the connecting loops considering the proximity of the
three residues. As supporting evidence, in the crystal structure of
highly homologous genotype 1a HCV RNA helicase which was determined
very recently, two molecules in the asymmetric unit displaying 3-4°
rigid body rotation of the RNA binding domain with respect to each
other undergo a small hinge bending motion of the two loops (33). The
domain movement appears intrinsically small due to the presence of the
two antiparallel
-strands which link the RNA binding and the helical
domain. The "structured" strands, interacting heavily with the
helical domain, are unlikely to undergo an appreciable conformational
change. In the crystal structure of genotype 1a HCV RNA helicase, the
-strands of the two molecules in the asymmetric unit show a
negligible twist with respect to each other. Consistently, despite the
completely different crystal packing, the RNA helicase structure
presented here (in the space group P3121) does not exhibit
any noticeable closure or opening of the interdomain cleft compared
with the structure of genotype 1a HCV RNA helicase (in the space group
P212121).
RNA Binding Motif--
The RNA binding motif,
Q460RRGRTGRGRRG sequence, shows unusually high
occurrence of glycine and arginine. The first three residues constitute
the end of an
-helix, and the rest of the residues forms a loop
structure (Fig. 1, 4). The high
occurrence of glycine on the loop structure strongly suggest that the
sequence can easily undergo a conformational change necessary for the
alignment of the arginine residues on the loop structure in favorable
contacts with the phosphate backbone of RNA. It was noted that the side chains of the most conserved residues, Gln460,
Arg461, Arg464, and Arg467, point
to the interdomain cleft with an ~7 Å spacing (Fig. 4).

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Fig. 4.
The conformation of the RNA binding motif of
HCV RNA helicase. The backbone atoms are in
yellow-brown and the side chains of highly conserved
residues (Gln460, Arg461, Gly463,
Arg464, Gly466, Arg467 and
Gly471) are in green, and the other residues are
in violet. The side chains of the conserved residues,
Gln460, Arg461, Arg464, and
Arg467, point to the interdomain cleft and are exposed to
solvent.
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Putative Functional Dimeric Model--
Entrapment of substrates
within protein structures has been observed as a common theme for
several proteins which possess a processivity in the interaction with
nucleic acids, including
subunit of E. coli DNA
polymerase III (34) and
-exonuclease (35). A functional oligomeric
state of HCV helicase is not known but has been proposed equivocally as
a monomer or a dimer (36). We examined crystal packing interfaces in
the crystal structure and found that a symmetry-related monomer-monomer
interaction could reflect interfaces of a functional form of the RNA
helicase. The interfaces are formed by the interactions between the
NTPase and the RNA binding domains of the two molecules and represent the most extensive crystal packing interactions (Fig.
5). The interactions screen a total of
144 Å2 surface area of one molecule. In the middle of the
dimer a channel is found that is helical in shape with a vertical
length of about 29 Å and a horizontal length of about 10 Å on the
front side and 12 Å on the back side. The RNA binding motif of each
molecule is the major part shaping the surface of the channel.

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Fig. 5.
A dimer interface which exhibits the most
extensive crystal packing interaction in the crystals of HCV RNA
helicase. A channel is found between the two symmetry-related
molecules through which ssRNA is able to pass. A crystallographic
2-fold symmetry axis is roughly perpendicular to the figure. The
interdomain cleft is predominately negatively charged (red).
A putative active dimeric form is proposed to be similar in
conformation to the symmetric dimer shown here (see text).
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It was possible to model a canonic RNA (37) into the interdomain cleft
of one molecule and continuously through the channel at the dimer
interface with the 3' end hanging out of the channel. In the modeling
experiment, the phosphate backbone of ssRNA was brought into contact
with the RNA binding motifs of the two molecules with slight dihedral
angle changes of the phosphate backbone. This was easily achieved
because both the two RNA binding motifs and the two interacting
phosphate groups are separated by ~17 Å corresponding to a half-turn
of RNA (Fig. 6). In this binding mode,
one molecule of the RNA helicase interacts with ssRNA extensively, whereas the other molecule reacts less extensively using the RNA binding motif only. The phosphate backbone of the bound ssRNA, interacting with the RNA binding motifs at the channel, appears as a
"glue" which stabilize the dimeric structure. Similar observations were made for the dimer or the trimer of the DNA polymerase
subunit, both of which are supposed to be stabilized by dsDNA entrapped
in the middle of the oligomeric structures (34, 38). It was noted that
the interdomain cleft is slightly wider for the ssRNA modeled in the
interdomain cleft. A small rotation of the RNA binding domain toward
the NTPase and the helical domain is expected for an induced fit of
ssRNA as a result of the hinge bending motion. It is known that ssRNAs
increase the NTPase activity of HCV RNA helicase up to 27-fold (36),
whereas dsRNAs do not. The activity increase is likely due to the ssRNA
binding to the interdomain cleft, which may also trigger some
conformational change at the active site of the enzyme. In this regard,
the RNA helicase with a bound ssRNA at the interdomain cleft can be
considered as an activated molecule, whereas the RNA helicase without a
bound ssRNA can be considered as a resting molecule. It is not known whether the RNA and the NTP binding to the enzyme are sequential or
random. Our modeling experiment, which shows that the bound ssRNA dose
not block the NTP binding site, cannot distinguish the two. Whether it
is sequential or random, ATP hydrolysis would occur mainly on the
activated molecule. Because of the expected small conformational change
upon the ssRNA binding at the interdomain cleft, the asymmetric
putative functional dimeric form composed of the activated molecule and
the resting molecule would be slightly different from the symmetric
dimer composed of the two resting molecules presented here. The
dimer interface in the crystal structure does not involve any specific
interaction such as ion pairs between charged amino acids. Thus, the
slight rotation of the RNA binding domain at the dimer interface
could easily occur upon the induced conformational change by the ssRNA
binding.

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Fig. 6.
Modeling of single stranded portion of RNA
into the interdomain cleft and through the channel between the two
molecules of the symmetric dimer shown in Fig. 5. In the modeling,
a canonic RNA was used, and the phosphate backbone of the ssRNA was
brought into contact with the RNA binding motifs of the dimer with
slight changes of the backbone dihedral angles of the ssRNA. The top
portion of the RNA binding domain facing the channel is the RNA binding
motif. The negative electrostatic potential of the interdomain cleft
suggests that it interacts with bases of ssRNA. This was accounted for
in the modeling experiment, but it was not necessary to change the
phosphate backbone dihedral angles.
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Mechanism of Duplex Unwinding--
With this putative functional
dimeric model, we propose a mechanism of processive unwinding of duplex
RNA coupled with the NTP hydrolysis by HCV and related RNA helicases.
ATP decreases the affinity of HCV RNA helicase for deoxyuracil 18 mer
by 95% (36), indicating that the ATP hydrolysis results in the
dissociation of RNA from the enzyme. Based on this observation, it can
be hypothesized that the NTP hydrolysis causes a hinge bending motion
which transforms the activated conformation of the enzyme to the
resting conformation concomitant with the detachment of the bound ssRNA
from the interdomain cleft. In the model presented here, the detached
ssRNA can be closer to and bound by the interdomain cleft of the
resting molecule of the putative functional dimer. This can be
described as a rotation of the dimer relative to the bound ssRNA (Fig.
7). In order for the dimer to translocate
on the ssRNA by the rotational motion, a rotation axis should be toward
the 5' end of the bound RNA relative to the "pseudo-" 2-fold
symmetry axis of the functional dimer. In the structure of the dimer,
the front part of the RNA binding motif of one molecule is below the
2-fold symmetry axis (Fig. 6). It was shown that individual alanine
substitutions of the conserved arginine residues in the RNA binding
motif in vaccinia virus RNA helicase cause severe defects in RNA
unwinding with slight reduction in RNA binding affinity (21). This
mutational study led to the conclusion that the motif must play an
essential role in the helicase mechanism. The structural observation
and the biochemical data suggest that a front part of the RNA binding motif serves as the pivoting region for the rotation in the context of
the proposed model. About 60° rotation of the dimer along an axis
passing the front part of the RNA binding motif of the activated molecule brings the resting molecule into contact with the ssRNA and
results in a translocation of the dimer toward the 5' direction as
shown in Fig. 7. After this one cycle of the rotation and translation the conformations between the activated molecule and the resting molecule are exchanged, reproducing the same RNA binding mode of the
functional dimer. Repeated cycles of the rotation and translation along
the ssRNA containing the 3' overhang can be described as "descending
molecular see-saw" motion (Fig. 7). Since the size of the interdomain
cleft is adequate only for ssRNA, duplex unwinding by the disruption of
base pairings should occur at the ssRNA and dsRNA junction. Required
energy can be provided by the favorable interaction between the
interdomain cleft and the ssRNA. The translocation of the dimer along
the ssRNA is the process of duplex unwinding because one strand of RNA
passing through the channel is separated from the other strand hanging
out of the dimer (Fig. 6, 7). The step size of duplex unwinding and
translocation along the ssRNA is about 5 nucleotides when the dimer
rotates along an axis at the front part of the RNA binding motif (about
Arg462). This coincides with the step size of UvrD helicase
obtained by kinetic measurement (39).

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Fig. 7.
A side view of "descending molecular
see-saw" model for the translocation of HCV RNA helicase along
ssRNA. The dimer (left) translocates along the ssRNA
(right) by a rotation of about 60° (with respect to the
axis of the RNA double helix) along an axis passing the front part of
RNA binding motif. The right figure represents a
translocation of the dimer by a half-turn of RNA with respect to the
location of the dimer on ssRNA in the left figure. For clear
presentation of the translational motion, the two figures are shown
with orientations different by 180° with respect to each step. Figs.
1, 3, and 5 were produced using the program MOLSCRIPT, Fig. 2 using the
program O, Fig. 4 using the program GRASP, and Figs. 6 and 7 using the
program QUANTA.
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The proposed functional dimeric model satisfies the previously observed
properties of the enzyme. First, the model requires a 3' overhang
region of RNA for the initial binding of one molecule the RNA and for
the formation of the putative active dimer (Fig. 8). This explains the requirement of an
at least 11-base-long single stranded portion of the RNA substrates for
the helicase function (36). It is the minimum length that spans the
interdomain cleft and the channel of the dimer (Fig. 6). Second, the
functional dimer moves along the ssRNA containing 3' overhang in the 3'
to 5' direction as previously observed (40). It was not possible to
insert ssRNA into the dimeric structure in the 5' to 3' direction simply due to severe steric clashes. Third, the model requires the
pivoting motion around the RNA binding motif for the duplex unwinding.
This explains that the RNA binding motif is necessary not only for the
binding of RNA but also more importantly for the unwinding activity of
the enzyme. Fourth, since the functional dimer translocates along only
one strand of nucleic acids in this model, the dimer is able to unwind
dsRNA, dsDNA, and DNA/RNA duplex as previously known. Besides, since
the dimer uses only the front side of the molecules for the duplex
unwinding and the flexible N terminus is located at the back side of
the molecule, the protease domain linked to the helicase domain in NS3
would not interfere the movement of NS3 along RNA substrates in
vivo.

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Fig. 8.
Schematic drawings for the proposed mechanism
of processive duplex unwinding by HCV RNA helicase. A, a
helicase molecule ( ) binds the single stranded portion of
RNA. B, subsequently, a functional dimer is formed by the
binding of another helicase molecule ( ). The ssRNA bound
to the interdomain cleft of is proposed to induce a
small conformational change which increases the NTP hydrolysis
activity. The dimer is stabilized by the interaction of ssRNA with the
RNA binding motifs of and . C,
the NTP hydrolysis by in B results in the
detachment of the ssRNA and a rigid body rotation of the dimer along an
axis at the RNA binding motif of . As a result, the dimer
translocates along the ssRNA in the 5' direction, and the interdomain
cleft of binds the other portion of the ssRNA.
D, the dimer reaches the junction of ssRNA and dsRNA by
repeated cycles of the translocation. E, in the same manner,
the dimer translocates along the same strand of RNA. Energy required
for the disruption of base pairings can be supplied by favorable
interactions between the interdomain cleft and the ssRNA. One strand
passing through the channel at the dimer interface is separated from
the other strand hanging out of the dimer.
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The proposed model is analogous in concept to the "rotary-type"
mechanism of F1-ATPase. It is believed that a 120°
rotation of
subunit of F1-ATPase induces sequential
conformational changes of the
3
3
subunits. Each subunit alternates among three conformations, ADP and
Pi bound, ATP bound, and none-bound (41). Like the
subunit of F1-ATPase, the bound ssRNA results in the
asymmetric functional dimer, each molecule of which alternates between
the activated and the resting conformation. Interestingly, it was proposed recently that hexameric T7 DNA helicase encircles only one
strand of DNA (42), as does the functional dimeric model proposed here.
In conclusion, the "descending molecular see-saw" model is
presented consistent with the previously observed biochemical data for
the RNA helicases. The model provides a plausible framework explaining
how the enzymes achieve the duplex unwinding and the translocation
along nucleic acids in a processive manner.