Structure of the three-way helical junction of the hepatitis
C virus IRES element
JONATHAN OUELLET, SONYA MELCHER, ASIF IQBAL, YILIANG DING,1and DAVID M.J. LILLEY
Cancer Research UK Nucleic Acid Structure Research Group, MSI/WTB Complex, The University of Dundee, Dundee DD1 5EH,
The hepatitis C virus internal ribosome entry site (IRES) element contains a three-way junction that is important in the overall
RNA conformation, and for its role in the internal initiation of translation. The junction also illustrates some important
conformational principles in the folding of three-way helical junctions. It is formally a 3HS4junction, with the possibility of two
alternative stacking conformers. However, in principle, the junction can also undergo two steps of branch migration that would
form 2HS1HS3and 2HS2HS2junctions. Comparative gel electrophoresis and ensemble fluorescence resonance energy transfer
(FRET) studies show that the junction is induced to fold by the presence of Mg2+ions in low micromolar concentrations, and
suggest that the structure adopted is based on coaxial stacking of the two helices that do not terminate in a hairpin loop (i.e.,
helix IIId). Single-molecule FRET studies confirm this conclusion, and indicate that there is no minor conformer present based
on an alternative choice of helical stacking partners. Moreover, analysis of single-molecule FRET data at an 8-msec resolution
failed to reveal evidence for structural transitions. It seems probable that this junction adopts a single conformation as a unique
and stable fold.
Keywords: RNA structure; RNA junctions; translation; FRET; single-molecule fluorescence
The internal ribosome entry site (IRES) of the hepatitis
C virus (HCV) binds eIF3 and the small ribosomal sub-
unit, allowing internal initiation of translation to occur
independently of a capped 59 terminus (Tsukiyama-Kohara
et al. 1992; Wang et al. 1993; Sizova et al. 1998). This 341-
nucleotide (nt) RNA is found upstream of a gene encoding
a large polyprotein. The HCV IRES RNA is highly con-
served, and adopts a folded structure in the presence of di-
valent metal ions (Kieft et al. 1999; Spahn et al. 2001). The
RNA has a complex secondary structure (Brown et al. 1992;
Reynolds et al. 1995; Rijnbrand et al. 1995; Honda et al.
1996), which includes a number of helical junctions, shown
schematically in Figure 1A. Domain III includes a prom-
inent four-way junction. Using the IUB nomenclature of
helical junctions (Lilley et al. 1995), this junction is defined
as 2HS22HS1, indicating the number of helical (H) and
linking segments (S) sequentially around the branchpoint.
There is also a less well-defined junction involving helices
IIIe and IIIf, and a three-way junction that includes helix
IIId (Fig. 1B). Elements of the structure have been de-
termined (Klinck et al. 2000; Lukavsky et al. 2000; Kieft et al.
2002; Lukavsky et al. 2003). We have previously studied the
structure and dynamic properties of the 2HS22HS1four-way
junction. The structure of a related construct was solved by
X-ray crystallography (Kieft et al. 2002), but we found that
this junction was either a stacked cross with axes perpen-
dicular or a dynamic structure oscillating between parallel
and anti-parallel stacked structures (Melcher et al. 2003).
Interestingly, an anti-parallel conformation was suggested by
a single-particle cryo-electron microscopy (cryo-EM) study
(Boehringer et al. 2005), whereas a parallel conformation
was found in the crystal structure (Kieft et al. 2002).
The three-way junction has not been the subject of ex-
tensive study, either structurally or functionally. The struc-
ture of helix IIId has been studied by nuclear magnetic
resonance (NMR) (Klinck et al. 2000; Lukavsky et al. 2000),
have been analyzed. To date, there has been no study of the
1Present address: Department of Chemistry, Penn State University,
University Park, PA 16802, USA.
Reprint requests to: David M.J. Lilley, Cancer Research UK Nucleic
Acid Structure Research Group, MSI/WTB Complex, The University of
Dundee, Dundee DD1 5EH, United Kingdom; e-mail: d.m.j.lilley@dundee.
ac.uk; fax: +44-1382-345893.
Article published online ahead of print. Article and publication date are at
RNA (2010), 16:1597–1609. Published by Cold Spring Harbor Laboratory Press. Copyright ? 2010 RNA Society.
it has been demonstrated that the apical GGG is critical for
for binding by the 40S ribosomal subunit. Since the confor-
mation of the junction will likely determine the presentation
of this loop to the ribosome, we expect that the junction will
be critical to IRES function.
Helical junctions are a major structure determinant of
complex RNA species (Lilley 2000). Their geometry orients
helices relative to one another, allowing long-range tertiary
contacts to occur. For example, inspection of the structures
of the 16S and 23S rRNA species within the ribosome reveals
many helical junctions (Ban et al. 2000; Wimberly et al.
2000). The structure of some autonomously folding smaller
RNA species can be almost entirely determined by compo-
nent junctions (Murchie et al. 1998; Walter et al. 1998), as
exemplified by the VS ribozyme where the structural core
of the ribozyme comprises five helical segments related by
two three-way junctions (Lafontaine et al. 2002; Lipfert et al.
of ribozymes, such as the hammerhead ribozyme (Martick
found in a number of riboswitches (Serganov et al. 2004,
2008; Garst et al. 2008). Three-way junctions are especially
common in functional RNA species of all sizes. In the
sections that follow we set out a new analysis of the structure
and topology of three-way junctions in general, and then
follow this with a discussion of the specific sequence of the
IRES three-way junction.
The helical arms of junctions in both DNA and RNA
have a strong propensity to undergo coaxial stacking when
electrostatic repulsion is minimized by the presence of metal
ions (Duckett et al. 1988, 1995; Orr et al. 1998; Walter et al.
1998; Lescoute and Westhof 2006; de la Pena et al. 2009). In
in this way, and the third must remain unstacked. The
simplest case is the perfectly base-paired 3H junction, where
the three interhelical connections each lack additional nu-
cleotides. However, 3H junctions are rare in natural RNA
prising one or more unpaired nucleotides, which we term
‘‘linking’’ nucleotides. These probably provide conforma-
by pairwise coaxial stacking. Examination of the structures
of three-way junctions observed in crystal structures of
RNA species(Lescoute andWesthof 2006)indicates astrong
propensity of junctions fold in a way that minimizes the
number of linking nucleotides between the stacked helices.
This is a reliable predictor of structure in natural three-way
junctions. A junction with two coaxially stacked helices is
depicted schematically in Figure 2A. It should be noted that
all three component strands are structurally distinct. One
strand runs the length of the stacked helices—we call this
strand ‘‘continuous’’ (con). The other two pass between the
in a 59-to-39 direction—we call this ‘‘exit’’ (ex). The other
In a junction that has a single linking segment, the linker (L)
can be located on either the ex strand (termed the Lex form),
to the Westhof structural families A and C, respectively
(Lescoute and Westhof 2006). It is important to note that
because the ex and en strands are inequivalent, the Lex and
Len structures are also inequivalent (unlike the two stacking
conformers of a four-way junction), and are likely to have
different stabilities. Where there are two linkers of equal
length we term this ‘‘equal’’ (Leq); this corresponds to the
Westhof family B (Lescoute and Westhof 2006).
The orientation of the third helix in the plane of the three
helices can also be specified with respect to the direction of
axes of the ex strand subtend an acute angle, or it can be
directed toward the 59 end, in which case it is the en strand
could be approximately perpendicular to the axis of the
FIGURE 1. The hepatitis C virus IRES. (A) Schematic of the secondary
structure of the IRES. The three-way helical junction studied here is
circled. (B) The local sequence around the three-way junction contain-
ing helix IIId. (C) The sequence of the three-way junction in the form
studied here. The junction comprises three strands (c, d, and e) and the
arms are labeled C, D, and E as shown. The loop and non-Watson–
Crick pairs have been removed from helix IIId to create a simple helix
D for the majority of this study. (D) A form of branch migration creates
three possible secondary structures at the junction.
Ouellet et al.
RNA, Vol. 16, No. 8
In the present study we have examined the HCV IRES
three-way junction that includes helix IIId. Since the
terminal loop of helix IIId binds to the 40S ribosomal
subunit (Kolupaeva et al. 2000; Kieft et al. 2001; Boehringer
et al. 2005), and certain mutations in the loop and internal
bulge can abolish HCV IRES binding (Jubin et al. 2000;
Klinck et al. 2000; Kieft et al. 2001), the structure of the
junction and the resulting trajectory of helix D are likely to
be important to the function of the IRES element. For this
work we have named the component helices C (the upper
arm from which extend helices IIIa, IIIb and IIIc), D (helix
IIId), and E (the lower arm that includes IIIe and IIIf) (Fig.
1C). The base pairing is conventionally drawn in the form
of a 3HS4junction. However, we note that the base pairing
could be rearranged slightly to create either 2HS1HS3or
undergo two steps of branch migration, with the 3HS4and
2HS2HS2junctions as the extremes. These three forms are
unlikely to be of equal free energy, but in the absence of
experimental data, we cannot predict which might be the
more stable. However, the secondary structure will have
a major influence on the fold adopted by the junction.
The 3HS4structure has a single linking segment of 4 nt,
and two connections with zero length linkers. There are,
therefore, two possible structures that might be adopted
in which either helices C and E, or helices D and C, are
coaxially stacked (Fig. 2C). Note that these are not equiv-
alent, as they are Len and Lex structures, respectively. Indeed,
Lescoute and Westhof (2006) speculated that the structure
of this junction might fall into either family A or C on the
basis of the 3HS4structure. However, if the junction adopts
either the 2HS1HS3or the 2HS2HS2secondary structure,
there would be two linking segments, and thus only one
way of stacking two arms without a linker. In this structure
helices C and E are coaxially stacked. The 2HS1HS3junction
is a Len structure, while the 2HS2HS2form is a Leq structure
From this analysis either secondary structure could
adopt the structure based on C upon E coaxial stacking.
However, it is likely that the orientation of the D arm (and
thus its interaction with the ribosome) would depend on
which secondary structure is formed. In this study we have
examined the conformation of the IRES junction using a
combination of comparativegelelectrophoresis and fluores-
cence resonance energy transfer (FRET) in both ensemble
steady-state and single-molecule mode. It emerges that in
addition to its functional significance, the IRES junction is
an interesting test of the conformational principles of three-
way helical branch points, requiring a consideration of both
secondary and three-dimensional structure.
Analysis of interhelical angles in the IRES
We have studied the structure of the three-way junction
shown in Figure 1B. The component helices of the junction
are Watson–Crick paired for at least three base pairs ex-
tending outward from the point of strand exchange. Helix
IIId contains a number of formal non-Watson–Crick pairs,
and a small internal loop; however, an NMR structure
showed this to be essentially helical without major axial
et al. 2000). For the major part of this analysis we have
therefore retained the central sequence of the junction and
extended its arms with perfectly paired helices (Fig. 1C),
we have named the helical arms C, D, and E as shown, where
helix D is equivalent to helix IIId in the complete IRES. The
arm in which the 59 terminus is located. This junction has
FIGURE 2. The structurally distinct strands of three-way junctions
with formally unpaired linking segments, and possible structures for
the IRES junction. (A) The component strands of a three-way junction
with two coaxially stacked helices are distinct. The con strand turns
about the shared axis of the stacked helices, while the en and ex strands
have their 59 and 39 termini in the nonstacked helix. (B) Three possible
forms of a junction with unpaired RNA linking the unstacked helix to
the coaxially stacked pair of helices. If the linker (or longer linker where
there are two) lies on the en or ex strand, we term the structures Len or
Lex, respectively, while if there are two linking segments of equal length
we call it Leq. (C) In principle the three-way junction of the IRES might
adopt alternative secondary structures, so there are a number of struc-
tures possible. The 3HS4secondary structure has a single segment link-
ing the D and E arms, and could therefore adopt either Len (C-on-E
stacking) or Lex (D-on-C stacking) structures in principle, or both in
equilibrium. The 2HS1HS3or 2HS2HS2structures would be expected to
fold by C-on-E stacking, forming Len and Leq structures, respectively.
A three-way junction in the HCV IRES
been analyzed using comparative gel electrophoresis and
Analysis of interhelical angles in the IRES three-way
junction using comparative gel electrophoresis
Comparative gel electrophoresis provides a simple yet pow-
erful way to examine the global conformation of a helical
junction in RNA under a variety of conditions (Lilley 2008,
2009a). The IRES junction was constructed from three
separate strands (each generated by transcription from
a DNA template), and prepared in different forms compris-
ing the three possible variants with two long (40 base pairs
[bp]) arms and one shortened (10 bp) arm. The species are
arms, and a shortened arm E. The relative electrophoretic
mobility in polyacrylamide gels of the three long–short arm
species reflects the angle subtended between the two long
The three species of the IRES junction have been elec-
trophoresed in 10% polyacrylamide gels under a variety of
ionic conditions (Fig. 3). In the absence of added metal
ions (with 2 mM EDTA to chelate any traces of metal ions),
the three species migrate with relative rates of DE > CE >
CD. This indicates that the largest angle contains the for-
mally unpaired CUUG sequence, suggesting a conformation
with an open center. The smallest angle is subtended be-
tween the C and D arms. Upon addition of metal ions the
pattern of electrophoretic mobility is significantly changed,
with relative rates CE > DE > CD. Thus, in common with
many helical junctions, the IRES three-way junction un-
dergoes a metal ion-induced change in conformation. The
fast mobility of the CE species would be consistent with
a predominant species present in a solution whose struc-
ture is based upon coaxial stacking of the C and E helices,
with the e strand passing continuously between the two
helices through the junction. The relative mobilities of the
remaining species indicated that helix D is directed into the
same quadrant as helix C. The global structure is apparently
closely similar in 1 mM Mg2+and 25 mM Na+ions, sug-
gesting that specific ion binding may not be required to
fold the junction. The same global geometry is also found
in the presence of the trivalent ion [Co(NH3)6]3+.
Analysis of relative end-to-end distances of the arms
of the IRES three-way junction using steady-state
fluorescence resonance energy transfer
FRET is an alternative way to analyze the global structure
of helical junctions in solution, based on energy transfer
between donor and acceptor fluorophores attached to the
ends of selected pairs of helical arms (Lilley 2009b). Effi-
ciency of the process depends inversely on the sixth power
of the distance between the fluorophores (Fo ¨rster 1948).
The IRES junction was constructed with three arms, each
comprised of 12 bp (when drawn with 3HS4secondary
structure), labeled with fluorescein donor and Cy3 acceptor
attached via the 59-termini of different strands (Fig. 4).
Fluorescein attached at the 59-terminus is relatively mobile
(Norman et al. 2000). This minimizes the complications of
fluorophore orientation, so that a simple interpretation
based on an inverse relationship between FRET efficiency
and distance is unlikely to be misleading. Three such spe-
cies were constructed, corresponding to the vectors (named
according to the arms carrying the donor and acceptor, in
that order) CD, CE, and DE.
FRET efficiency for the three vectors was measured in the
steady state as a function of Mg2+ion concentration (Fig.
4). This clearly confirms that the structure of the IRES junc-
tion depends on the metal ion concentration. The relative
interfluorophore distances at low ionic concentration are
DE > CE > CD, fully consistent with the global structure
deduced from comparative gel electrophoresis. Further-
more, the relative distances in the presence of $1 mM
FIGURE 3. Comparative gel electrophoresis of the conformation
adopted by the three-way junction of the HCV IRES in different ionic
conditions. The junction studied had a simplified helix D, with the
central sequence shown in Figure 1C. Three forms of the junction,
each with a different helical arm shortened, are electrophoresed in
adjacent tracks of a 10% polyacrylamide gel in the presence of 90 mM
Tris-borate (pH 8.3) with the indicated metal ions (or EDTA to
chelate metal ions). The long–short-arm junction species are named
according to the two long arms. An interpretation of the structures
adopted is shown on the right, together with the anticipated structures
and mobilities of the long–short-arm junction species. In the presence
of EDTA the slow-intermediate-fast pattern is interpreted in terms of
the extended structure, while the slow-fast-intermediate pattern ob-
served in the presence of metal ions is interpreted in terms of a struc-
ture in which helices C and E are coaxially stacked, with an acute angle
subtended between helices C and D.
Ouellet et al.
RNA, Vol. 16, No. 8
Mg2+(CE > DE > CD) are also consistent with the structure
of the predominant species from the electrophoretic exper-
iments in the presence of metal ions. The titrations can be
fitted to a two state transition in which the junction folds in
the CE and DE vectors give half-magnesium concentrations
of [Mg2+]1/2= 4 and 7 mM, respectively. Hill coefficients of
0.52 for both vectors indicate an anti-cooperative process
under these conditions, consistent with folding induced by
the diffuse interaction of metal ions.
Analysis of the conformation population of the IRES
three-way junction using single-molecule FRET
Although electrophoresis and steady-state FRET indicate
a predominant conformation that is probably based upon
coaxial stacking of C on Earms,these experimentscould not
exclude a small population of an alternative conformer in
equilibrium. We therefore searched for such a conformation
and CD vectors, using Cy3 and Cy5 as donor and acceptor
fluorophores, respectively (Fig. 5). In addition, we prepared
an identically labeled 24-bp duplex species by hybridization
that are coaxially stacked. All species were studied as single
junction molecules encapsulated in phospholipid vesicles in
the presence of 10 mM Mg2+ions, using prism-based total
internal reflection fluorescence microscopy. Time traces of
donor and acceptor intensities were recorded at time reso-
lutions between 100 and 8msec, for hundredsof single junc-
tions, and histograms of FRET efficiency were calculated
after filtering anomalous data (see Materials and Methods).
EFREThistograms of data at 100-msec resolution for the
junction and duplex species are presented in Figure 5. The
CD and CE vectors of the junction give distributions that
are well fitted by single Gaussian curves, with mean EFRET
values of 0.62 and 0.29, respectively (Table 1). These values
are qualitatively in good agreement with the model result-
ing from the electrophoresis and steady-state FRET data.
Moreover, the value for the CE vector is similar to that of
the simple CE duplex (EFRET= 0.32), as would be expected
if the C and E arms were coaxially stacked in the folded
junction. The widths of both distributions for the junction
species are not much greater than that for the duplex,
FIGURE 4. Analysis of metal ion-induced folding of the IRES three-
way junction by steady-state FRET measurements. The junction
shown in Figure 1C was prepared with fluorescein (donor) and Cy3
(acceptor) fluorophores attached to the 59-termini of selected helical
arms. These vectors are named by the labeled arms, in the order
donor–acceptor, so that the CD vector has fluorescein attached to
helix C and Cy3 to helix D. FRET efficiency was measured using the
acceptor normalization procedure as a function of Mg2+ion concen-
tration in 90 mM Tris-borate (pH 8.3), and plotted for the three
vectors CD (j), DE (s), and CE (d). The data have been fitted to
a two-state model of ion-induced folding (—).
FIGURE 5. Population distributions of FRET efficiencies for Cy3–
Cy5-labeled IRES junctions studied as single molecules. Except for
the different fluorophores, equivalent junction species as used in the
steady-state fluorescence experiments (Fig. 4) were studied. In addition,
a Cy3–Cy5-labeled duplex was prepared by hybridization of the junc-
tion e strand to its complement, to provide a model for coaxially
stacked C and E arms. The different species were encapsulated in phos-
pholipid vesicles in 10 mM Tris-HCl (pH 8.1), 50 mM NaCl, 10 mM
MgCl2, and imaging buffer (see Materials and Methods) and studied by
total internal reflection fluorescence microscopy. Hundreds of single
molecules were studied for each species, and fluorescent intensities at
Cy3 and Cy5 emission wavelengths recorded for a number of minutes
with a 100-msec resolution. Molecules with aberrant spectral properties
were rejected and FRET efficiencies calculated from the remainder.
These were plotted as the histograms shown, fitted to Gaussian dis-
tributions. Histograms are shown for the CD junction, the CE junction,
and the CE duplex. The measured mean EFRETand half-widths for each
distribution are presented in Table 1.
A three-way junction in the HCV IRES
indicating that the arms of the junction do not undergo
flexural motions of large amplitude. The data for the CD
vector has points that are distributed over a wider range of
from the smaller number of molecules analyzed compared
with the CE species.
of EFRET, i.e., donor–acceptor distance. In the histogram
for the CE vector there is no detectable peak at higher values
at EFRET= 0.3, but they are randomly distributed rather than
forming a distinct peak. Similar histograms were calculated
from the data recorded at a faster acquisition time. These
data provide no evidence for a minor conformation present
in solution under these conditions.
No conformational transitions have been detected
in the IRES three-way junction using
As a further way of seeking alternative conformations,
we examined the time records for many single junction
molecules, looking for anti-correlated changes in donor
and acceptor intensities that would indicate a transition
to a different structure. At 100-msec time resolution we can
follow donor and acceptor fluorescent intensities over 200
sec or more before photobleaching. Examples of CD and
CE vectors are shown in Figure 6, A and C, respectively,
with their corresponding FRET efficiency histograms in B
and D, respectively. There is no evidence for any structural
transition involving a change of FRET efficiency in these
traces, and we found very few examples of a transition in
the hundreds of time traces examined.
We also sought short-lived conformational states by
examining time traces recorded in the presence of 10 mM
MgCl2at 8-msec time resolution; the highest possible using
our EMCCD camera. A 1-sec section of a trace for the CD
vector is expanded in Figure 6E. No evidence in this trace
(or in many others studied) can be seen for anti-correlation
of Cy3 and Cy5 intensities. This is further demonstrated by
calculating a cross-correlation function from a randomly
chosen 4 sec of data (Fig. 6F). These data show no indi-
cation of a decaying anti-correlation, fitting a linear, hori-
zontal function passing through zero. A small subset of
junction species (three molecules out of a total of 323)
underwent multiple slow transitions between states of
different FRET efficiencies; these molecules are clearly of
a different character from the majority and are likely to be
misfolded or mishybridized junctions.
In summary, using faster acquisition and cross-correla-
tion analysis we have found no evidence for an additional
conformation of the junction.
Studying the secondary structure of the IRES
three-way junction using in-line probing
In view of the uncertainty with regard to the secondary
structure adopted by the IRES three-way junction, we per-
formed in-line probing experiments in an attempt to
identify conformationally flexible nucleotides within the
core. In this approach, radioactively [59-32P]-labeled RNA
is subjected to a prolonged incubation in the presence
of buffer and Mg2+ions. Where the backbone is relatively
flexible, the RNA can sample a conformation in which the
29-OH may carry out an in-line nucleophilic attack on
the 39-phosphate, but this is hindered in more rigid parts of
the molecule, including duplex regions. Using transcription
by T7 RNA polymerase, we prepared two constructs both
of which comprised a single strand of RNA, with terminal
hairpin loops on either the C and E or the D and E helices
(Fig. 7) in order to maximize the resolution of the se-
quences of interest by gel electrophoresis.
As expected, the data reveal that the nucleotides of the
loops are flexible. But interestingly, this analysis shows
quite clearly that there are two regions of flexibility in the
core of the junction. The center of the strand linking the C
and E helices (i.e., what would be the e strand if the junction
comprised three separate strands) exhibits no detectable
reactivity, consistent with the coaxial stacking of the C and E
arms. However, the remaining two linking regions do ex-
hibit reactivity, with a single point of cleavage between the C
and D arms (equivalent to the c strand), clearly visible in
Figure 7A, and four consecutive cleavages between D and E
(equivalent to the d strand), seen most clearly in Figure 7B.
These data suggest that the predominant form of the junc-
tion is 2HS1HS3, although we do not believe that is con-
clusive. We considered repeating the analysis with junctions
in which the secondary structure is locked into place, but
this would require changes of sequence at the center of the
junction that might well alter the folding significantly. At
the present time the most probable conclusion from our
data is that the junction is the 2HS1HS3structure, but it
is possible that branch migration occurs that generates a
TABLE 1. Conformational populations for the different Cy3–Cy5
vectors constructed from the IRES junction with helix D in its
simplified and natural forms calculated from single-molecule FRET
Simplified helix D
Natural helix D
0.62 6 0.002
0.29 6 0.0005
0.32 6 0.0004
0.086 6 0.004
0.057 6 0.001
0.050 6 0.004
0.46 6 0.0006
0.30 6 0.0005
0.062 6 0.001
0.052 6 0.001
The population histograms of FRET efficiency (EFRET) were fitted to
Gaussian functions from which the mean EFRETand half-widths
Ouellet et al.
RNA, Vol. 16, No. 8
population of the other forms if this did not alter the global
folding of the junction.
Restoration of the natural secondary structure
of helix D does not significantly perturb
the structure of the junction
In the natural form of the junction the secondary structure
of helix D contains an internal loop and a number of non-
Watson–Crick base pairs after the third base pair from the
junction. These were removed from the junction studied
above to provide a regular helix to facilitate the analysis. An
NMR study of a stem–loop with the sequence of helix D
reveals it to adopt the general structure of an A-form helix
in broad terms, but in principle, nucleotides from the helix
might interact with those of the junction and alter its gross
structure or induce transitions between alternative struc-
tures. To test this possibility we repeated the single-
molecule analysis on a new form of the
junction in which the natural structure
was restored to arm D. The UUGGGU
terminal loop was replaced by four
additional base pairs, and the vectors
CD and CE were prepared by addition
of 59-linked Cy3 and Cy5 fluoro-
phores as before (Fig. 8A).
The results from the single-molecule
FRET analysis are qualitatively similar
to those from the junction with a regular
helix D. The histograms for the two vec-
tors both reveal a single peak of FRET
efficiency, with CD > CE (Fig. 8B). The
EFRET value for the CE vector (0.30)
(Table 1) is slightly greater than that
observed for the simplified junction
(0.29), but still very much in the range
expected for a coaxial C–E alignment.
The half-widths are also similar. The
structure of the natural junction is
therefore likely to be based on C-on-E
helix stacking. However, the EFRET of
the CD vector is significantly lower than
that for the simplified junction. This
might result from a reorientation of he-
lix D arising from new interactions with
unpaired bases in that helix. But there
are other potential origins for different
FRET efficiencies between the two vec-
tors. The D helix in the modified junc-
tion is longer than that of the simplified
junction by 3 bp, and the Cy5 position
is likely to be rotationally different be-
tween the two with consequent differ-
ences in the relative orientation of the
fluorophore transition moments, which
can significantly influence FRET efficiency (Iqbal et al.
2008a). Even if orientational effects were unimportant, the
FRET efficiency corresponds to a distance that is compa-
rable to the likely Fo ¨rster length R0, where a relatively small
change in distance can result in a significant change in
EFRET. As with the simplified junction, we observe no anti-
correlated changes in fluorescent intensity between donor
and acceptor for either vector (Fig. 8C), providing no
evidence for structural transitions in the junction.
The three-way junction of the HCV IRES element turns out
to be an interesting object lesson in the conformational
possibilities of RNA branch points. Like most nucleic acid
junctions, it requires the presence of metal ions to fold
from an extended form to a form based on pairwise coaxial
stacking of helical arms. The transition is induced by Mg2+
FIGURE 6. Seeking conformational transitions in single-junction molecules. The CD and CE
vectors (the same species used in Figure 5) were separately encapsulated in phospholipid
vesicles in 10 mM Tris-HCl (pH 8.1), 50 mM NaCl, 10 mM MgCl2, and imaging buffer, and
studied by total internal reflection microscopy. Representative long time records at 100-msec
time resolution are shown for the CD and CE vectors (A and C, respectively) up to the point at
which fluorophore photobleaching has occurred. Histograms of EFRETfor the individual CD
and CE molecules are also presented as plots B and D, respectively, for the unbleached sections
of the time records. Note that no transitions can be detected over these long time traces. The
same preparation of CD vector was also studied at 8-msec time resolution. A 1-sec section of
a time record is presented (E). Close examination of this (and many other traces not shown)
fails to reveal anti-correlation between Cy3 and Cy5 fluorescence intensity. This is confirmed
by performing a cross-correlation analysis on a 4-sec time record (F). The data fit a linear
function with no decay, passing through zero.
A three-way junction in the HCV IRES
ions in low micromolar concentrations, with a Hill coeffi-
cient suggestive of nonspecific binding. But the major in-
terest lies in the structure adopted in the presence of metal
Comparative gel electrophoresis and steady-state FRET
studies clearly indicate that in the presence of metal ions
the junction predominantly adopts a conformation in
which arms C and E could be colinear, with an acute angle
subtended between arms C and D. The similarity in FRET
efficiency between the CE vector of the junction measured
in single junction molecules, and a duplex made by hy-
bridizing the e strand to its complement, is consistent with
the coaxial stacking of the C and E arms in the folded
junction. The half-width of the length distribution for the
CE vector of the junction is only a little greater than that of
the duplex, indicating that the center of the junction is not
very flexible. The mean FRET efficiency is slightly lower for
the junction (EFRET = 0.29) compared with the perfect
duplex (EFRET= 0.32). While a rotation of the C and E arms
hinged about the center would shorten the end-to-end
distance and have a relatively small effect on fluorophore
orientation, there are two ways in which the efficiency might
be reduced compared with the duplex. First, if these arms
were unstacked and pulled apart to dislocate the junction,
this could increase the end-to-end distance in the junction.
Second, a rotation of the helical arms about their common
axis might lead to a change in k2that gives a lower EFRET.
It is known that Cy3 and Cy5 fluorophores may be stacked
onto the helical ends of nucleic acid duplexes (Norman et al.
2000; Iqbal et al. 2008b) resulting in significant modulation
of energy transfer due to the orientation of their transition
dipole moments (Iqbal et al. 2008a). However, the manner
of fluorophore attachment to the RNA is not the same as
was used in that study, so we cannot be certain about the
nature of the RNA–fluorophore interaction in the present
case. Both these effects might occur, each contributing to the
lower observed EFRETof the junction compared with the
If the junction significantly populated the 3HS4secondary
structure, it is possible that a subfraction would adopt an
alternative conformer in which D and C arms are stacked
(Fig. 2C). No minor fraction could be detected using single-
molecule FRET, nor were any transitions to such a species
observed in the hundreds of single molecules studied. If an
alternative conformer exists, it must have a lifetime that is
significantly shorter than 8 msec. However, such a fast ex-
change situation is unlikely. If a small population of the
D-on-C stacked conformer was in fast equilibrium with the
FIGURE 7. Analysis of the structural flexibility of nucleotides in the IRES three-way junction using in-line probing. Radioactively [59-32P]-
labeled RNA was incubated in 50 mM Tris-HCl (pH 8.3), 100 mM KCl, and 20 mM MgCl2for various times at 21°C, and analyzed by denaturing
gel electrophoresis and phosphorimaging. The junction was studied as a single strand by placing terminal loops onto two helices. In order to
maximize the resolution of the sequences of interest, two constructs were studied, each with a single 59-terminus in either helix D (A) or helix C
(B). Bands were assigned by reference to cleavage by ribonuclease T1 (A, lanes 1,8; B, lane 3) and a hydroxide cleavage ladder (A, lanes 2,7; B, lane
2). The junction with open helix D was subjected to in-line probing for 5 min, 12, 24, and 48 h (A, lanes 3–6, respectively), and the open C arm
form to a 48-h incubation (B, lane 1). The nucleotides were numbered according to Honda et al. (1999).
Ouellet et al.
RNA, Vol. 16, No. 8
majority C-on-E form, this would be expected to increase
the mean EFRETof the CE vector. But as we have discussed,
the measured value is actually lower than the corresponding
duplex, not higher. Thus if the junction has the 3HS4sec-
ondary structure, it must be strongly biased to the C-on-E
By contrast, if the junction has either a 2HS1HS3 or
2HS2HS2secondary structure the bias to the single C-on-E
stacking conformer is immediately explained, because the e
strand is the only one lacking a linker. The in-line probing
experiment data point toward the 2HS1HS3structure with
the identification of a single flexible nucleotide on the ex
strand c, multiple flexible nucleotides on the en strand d,
and absence of cleavage on the con strand e. However, this
indirect method cannot be regarded as conclusive proof of
the structure. If the predominant form is 2HS1HS3, then
the structure adopted is quite unusual. It is a Len structure,
in which the nonstacked D arm bends into the same quad-
rant as the C arm. In principle, the nonstacked might bend
either way, so that the linker-containing strand turns
through an angle that is greater or less than 90°. If we imag-
ine fusing a fourth helix at the position of the linker, these
would correspond to parallel or anti-parallel conformations
of the resulting four-way junction, re-
spectively. In Len junctions of known
structure the linker generally turns
through >90° (a pseudoparallel struc-
ture, where the ex and en strands cross
one another), including the hammer-
head ribozyme (Pley et al. 1994; Scott
et al. 1995), the VS ribozyme III–IV–V
junction (Lafontaine et al. 2002), as well
as various cases identified in the ribo-
some by Lescoute and Westhof (2006).
But in the 2HS1HS3secondary structure,
the shorter linker turns through <90°,
adopting the pseudo-anti-parallel struc-
ture. This seems to be without precedent
in the structural database; no 2HS1HS3
three-way junction appears in the RNA
On the other hand, if the junction has
ing junction is Leq. These tend to fall in-
to the Westhof family B structure, which
In summary, all the evidence indi-
cates that the three-way junction adopts
a rather stable fold, in a single stacking
conformer, fixing the trajectory of helix
IIId (helix D in this study) in the struc-
ture. It is possible that it undergoes
some limited branch migration that is
not detectable in our single-molecule
experiments, but no exchange with an
alternative stacking conformer has been detected. It is likely
that this structurally stable junction has been selected by
a requirement for this relatively small RNA to adopt a stable
structure that can mediate the interactions with the 40S
ribosomal subunit and the translational initiation factor
eIF3. While this structure may become distorted upon
binding to the ribosome, its stability suggests that it will
retain its conformation.
MATERIALS AND METHODS
RNA synthesis and preparation of junctions
RNA oligonucleotides were made by chemical synthesis, either
commercially using 29-ACE chemistry (Dharmacon) or using
t-BDMS phosphoramidite chemistry (Beaucage and Caruthers
1981), as described by Wilson et al. (2001). Fluorescein and Cy3
fluorophores were coupled to the oligonucleotides used in the
steady-state fluorescence measurements during synthesis. For
single-molecule studies all oligonucleotides were made with 59
aminolinkers, and Cy3 and Cy5 fluorophores were conjugated as
N-hydroxysuccinimide esters (Amersham Biosciences). All oligo-
nucleotides were purified by gel electrophoresis in polyacrylamide,
and recovered from gel fragments by electroelution or diffusion
FIGURE 8. Single-molecule FRET analysis of the three-way IRES junction with helix D of the
natural sequence. (A) The sequence of the junction studied. This is identical to that of the
simplified junction except for the restoration of the base pairing found in the natural junction.
However, the terminal loop was removed and the base pairing extended to form a stable helix
with a 59-terminus for fluorophore attachment. The junction is formally depicted in the 3HS4
secondary structure. (B) Population distributions of FRET efficiencies for Cy3–Cy5-labeled
vectors encapsulated in phospholipid vesicles in 10 mM Tris-HCl (pH 8.1), 50 mM NaCl, 10
mM MgCl2, and imaging buffer measured at 100-msec time resolution. The total number of
molecules included were 1693 and 860 for the CD and CE vectors, respectively. (C) A
representative 1-sec time record for the CD vector at 8-msec resolution (left). Four seconds of
data were used as input into a cross-correlation analysis (right). These data show no evidence
of structural transitions at this time resolution.
A three-way junction in the HCV IRES
in buffer followed by ethanol precipitation. Fluorescently labeled
RNA was subjected to further purification by reversed-phase
HPLC on a C18 column (Waters m-Bondapak) using an aceto-
nitrile gradient with an aqueous phase of 100 mM triethylammo-
nium acetate at pH 7.0.
Three-way junctions were constructed by mixing one Cy3-
labeled strand, one Cy5-labeled strand, and the remaining non-
labeled strand (each 300 pmol) in 90 mM Tris-borate (pH 7.0)
and 25 mM NaCl, and annealed by heating at 85°C and cooled
slowly to 4°C. The hybridized junctions were purified by electro-
phoresis in 20% polyacrylamide in 90 mM Tris-borate (pH 8.3)
and 25 mM NaCl, and recovered by electroelution and ethanol
Transcription of RNA
DNA templates were prepared by 30 cycles of PCR using KOD
Hot Start DNA polymerase (Novagen) and 1 mM of each over-
lapping oligonucleotide, with an annealing temperature of 66°C.
RNA was transcribed from 0.15 mM of PCR product in the
presence of 40 mM Tris-HCl (pH 8.0), 20 mM MgCl2, 2 mM
spermidine, 4 mM of each NTP (Roche), 0.1 U of pyrophospha-
tase (Sigma), 0.01% Triton X-100 (Sigma), and T7 RNA poly-
merase for 2.5 h at 37°C (Milligan et al. 1987). The RNA was
purified by electrophoresis in a 6% polyacrylamide gel containing
7 M urea. It was visualized by UV shadowing and recovered by
electroelution into 8 M ammonium acetate and ethanol pre-
cipitation. Purified RNA (6 pmol) was radioactively 59-32P-labeled
using T4 polynucleotide kinase (NEB) and 80 nM of [g-32P]-ATP
(6000 Ci/mmol; Perkin-Elmer) for 1 h at 37°C.
Comparative gel electrophoresis
Radioactively [59-32P]-labeled species were electrophoresed in 10%
polyacrylamide gels (29:1, acrylamide:bis) in 90 mM Tris-borate
(pH 8.3), plus added salts or 2 mM EDTA as required. Electro-
phoresis was performed at 120 V at room temperature, with
recirculation of the buffer at >1 l h?1. Gels were dried onto
Whatman 3MM paper, exposed to storage phosphor plates, and
imaged using a Fuji BAS-1500 PhosphorImager.
The sequences used for the electrophoretic experiments were:
c strand: 59-GCGCAAGCGACAGGAACCUCGAGGGAUCCGGC
d strand: 59-GCGCAAGCGACAGGAACCUCGAGAAGCUUCGC
e strand: 59-GCGCAAGCGACAGGAACCUCGAGUCUAGACGC
Further shortened strands were made in order to truncate selected
helical arms to 10 bp.
Steady-state fluorescence spectroscopy
Fluorescence spectra were recorded in 90 mM Tris-borate (pH
8.3) at 4°C using an SLM-Aminco 8100 fluorimeter with Phoenix
Electronics (ISS). Spectra were corrected for lamp fluctuations and
instrumental variations, and polarization artifacts were avoided by
setting excitation and emission polarizers crossed at 54.7°. Values
of FRET efficiency (EFRET) were measured using the acceptor nor-
malization method (Clegg 1992) implemented in MATLAB. FRET
efficiency as a function of metal ion concentration was analyzed
on the basis of a model in which the fraction of folded molecules
corresponds to a simple two-state model for ion-induced fold-
EFRET¼ E0þ DEFRETKAM
1 þ KAM
where E0is the FRET efficiency of the RNA in the absence of added
metal ions, DEFRETis the increase in FRET efficiency at saturating
metal ion concentration, [M] is the prevailing metal ion concen-
tration, KA is the apparent association constant for metal ion
binding, and n is a Hill coefficient. Data were fitted to this equation
by nonlinear regression. The Mg2+ion concentration at which the
transition is half complete ([Mg2+]1/2) is given by (1/KA)1/n.
The sequences used in the FRET analyses were:
c strand: 59-CCUUGACUGCUAGCCGAGUACAGG-39;
d strand: 59-CCUGUACUCGGCCUUGUGGUCUCAACGG-39; and
e strand: 59-CCGUUGAGACCAUAGCAGUCAAGG-39.
A duplex corresponding to coaxially stacked C and E arms was
made by hybridization of Cy5-labeled e strand to its complement
(e9 strand) 59-labeled with Cy3:
e9 strand: 59-CCUUGACUGCUAUGGUCUCAACGG-39.
Encapsulation of RNA junctions in phospholipid
vesicles for single-molecule studies
A 100:1 molar ratio of unmodified and biotinylated phospholipids
was prepared by evaporating a number of aliquots of a mixture
of 2.5 mg L-a-phosphatidylcholine and 35 mg 1,2-dipalmitoyl-
sn-glycero-3-phosphoethanolamine-N-(cap biotinyl) (Avanti Po-
lar Lipids, Inc.) from chloroform in a stream of argon. The
aliquots were hydrated in 250 mL of 50 mM NaCl, 10 mM Tris-
HCl (pH 8.1) (TN50 buffer) without (one aliquot, used for
surface coating of slides) or with an addition of 200 nM of a given
fluorescent RNA junction and 10 mM MgCl2, corresponding to
5 pmol of RNA in a final volume of 250 mL.
The RNA was encapsulated in phospholipid vesicles by repeated
extrusion through a polycarbonate membrane containing 200-nm
pores using a mini-extruder (both Avanti Polar Lipids, Inc.),
creating 200-nm diameter unilamellar vesicles (Boukobza et al.
2001; Okumus et al. 2004). The ratio of RNA to phospholipid
resulted in most vesicles being empty, and thus most encapsula-
tions involved a single RNA junction molecule. Slides were pre-
pared by injection of RNA-free phospholipid vesicles into a narrow
channel made between a quartz microscope slide and coverslip
No 1.5 (VWR international) using double-sided adhesive tape,
and left at 4°C for 1 h to allow supported bilayer formation. The
sample chamber was then washed with TN50 buffer, treated with
0.2 mg/mL neutravidin (Pierce) for 10 min, and washed. A 1/20th
dilution of a chosen encapsulated RNA was injected and allowed
to bind to the neutravidin for 15 min. Imaging was performed
under the same buffer conditions used for vesicle preparation
with an oxygen-scavenging system consisting of 1.6 mg/mL of
glucose oxidase, 0.2 mg/mL of catalase, 6% (w/v) glucose, and
Ouellet et al.
RNA, Vol. 16, No. 8
1 mM TROLOX (6-hydroxy-2,5,7,8-tetramethylchroman-2-car-
Total internal reflection single-molecule microscopy
Fluorescence intensities at donor and acceptor wavelengths were
acquired from single junction molecules encapsulated in phos-
pholipid vesicles using total internal reflection fluorescence micro-
scopy. The sample was mounted on the stage of an inverted micro-
scope (Olympus IX70) and excited via the evanescent field
generated by the total internal reflection of light from a solid
state 532-nm laser (Crystalaser) via a quartz prism. Fluorescent
emission was collected by a 1.2-numerical aperture 60X water
immersion objective lens (Olympus), and separated by a 645-nm
dichroic mirror (Chroma Technology) into the donor and the ac-
ceptor fluorescence. These were focused side by side into a back-
illuminated EMCCD camera (iXON, Andor Technology) (Ha
2001). Hundreds of molecules could be recorded simultaneously
using an image area of 8.2 3 8.2 mm (512 3 512 active pixels).
Data were acquired using software written in Visual C++ (Micro-
soft), where each frame had a duration of 100 msec (10 frames
sec?1) for the population histograms and 8 msec (125 frames
sec?1) for the cross-correlation analysis. The highest acquisition
rate of the camera is 33 msec, but faster rates can be attained by
subdivision of the active number of pixels (16 msec/frame can be
achieved dividing the chip by 2 and up to 8 msec/frame using one
quarter of the chip).
Measurements were performed at room temperature. Single-
molecule FRET efficiency after background correction was ap-
proximated by EFRET = IA/(IA+ID), where IA and ID are the
fluorescence intensities of the acceptor and donor, respectively.
Because the quantum yields and detection efficiencies of Cy3 and
Cy5 are very close, EFRETclosely matches the true efficiency of
energy transfer. However, the spectral overlap separation of Cy3
and Cy5 is not total and the 645-nm separation led to z10% of
Cy3 leakage into the Cy5 channel. This introduces an apparent
EFRET around 0.1 for a single active Cy3 fluorophore in the
absence of the Cy5 acceptor.
Data analysis was carried out using laboratory-written analysis
routines developed in MATLAB. Single-molecule FRET histo-
grams were obtained using the whole FRET trace, while the
population histograms were obtained by averaging frames 11–20
for every individual molecule after manually filtering photo-
bleaching and blinking effects. Filtering involved removal of time
traces in which (1) the fluorescence intensity of the acceptor was
lower than 50–55 (caused by the absence of Cy5 fluorescence); (2)
the total intensity was higher than 550–700 (indicating the
presence of multiple fluorophores); (3) absence of anti-correlation
between both fluorescence intensities during a blinking, dynamic,
or a photobleaching event; (4) the total intensity was irregular
over the length of the time-trace; and (5) multiple photobleaching
events from the same fluorophore.
Cross-correlation analysis was performed using a program
implemented in MATLAB. Records of donor and acceptor in-
tensities as a function of time for single-junction molecules were
analyzed using the following equation:
G t ð Þ ¼+ IDt ð Þ ? ID
??? IAt þ t
N ? +ID? IA
ð Þ ? IA
where ID and IA are mean donor and acceptor intensities, re-
spectively, over the whole data record, normalized by the number of
points summed (N). The function G(t) correlates the donor intensity
at time t with acceptor intensity at time increment t later.
In-line probing of nucleotide flexibility
Two forms of the IRES junction were generated by transcription
of a single RNA species, forming junctions with 59-termini in either
the C or D arms (Fig. 7):
Terminus in C helix: 59-GGGCAGUCAACUGAUGAGGCCGAAA
Terminus in D helix: 59-GGGCCGAGUACUGAUGAGGCCGAAA
During transcription, the hammerhead ribozyme (underlined)
underwent cleavage generating a homogenous 59-end at the IRES
junction RNA. These were separately subjected to the in-line pro-
bing analysis of Soukup and Breaker (1999). Approximately 0.1
pmol of [59-32P]-labeled RNA was incubated in 20 mL 50 mM Tris-
HCl (pH 8.3), 100 mM KCl, and 20 mM MgCl2between 1 min
and 48 h at 21°C. Aliquots were diluted with 0.5 vol of 100 mM
Tris-HCl (pH 7.5) and 20 mM EDTA in formamide (termination
buffer). Uniformly cleaved RNA was generated by heating 0.1
pmol [59-32P]-labeled RNA in 50 mM NaOH at 90°C for 20 sec
before addition of six volumes of termination buffer and placing
on ice. RNA bands were assigned by comparison with 0.1 pmol
[59-32P]-labeled RNA partial digested with 0.4 U of ribonuclease
T1 (which cleaves 39 to G nucleotides) in the buffer provided by
the manufacturer (Ambion). Products of cleavage were separated
by electrophoresis in 10% Long Ranger (Lonza) polyacrylamide
gels containing 7 M urea, 40% formamide, 90 mM Tris-borate
(pH 8.3), and 10 mM EDTA, dried onto Whatman 3MM paper,
and visualized by exposure to storage phosphor plates and phos-
We thank Dr. Tim Wilson for discussions, Dr. Carlos Penedo for
providing software, and Cancer Research UK for financial support
of these studies.
Received March 3, 2010; accepted May 4, 2010.
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