Structure of the three-way helical junction of the hepatitis C virus IRES element

Abstract

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 3HS(4) junction, with the possibility of two alternative stacking conformers. However, in principle, the junction can also undergo two steps of branch migration that would form 2HS(1)HS(3) and 2HS(2)HS(2) junctions. Comparative gel electrophoresis and ensemble fluorescence resonance energy transfer (FRET) studies show that the junction is induced to fold by the presence of Mg(2+) 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.

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 containing 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.

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 5′ and 3′ 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 structures possible. The 3HS₄ secondary structure has a single segment linking 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 2HS₁HS₃ or 2HS₂HS₂ structures would be expected to fold by C-on-E stacking, forming Len and Leq structures, respectively.

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 observed in the presence of metal ions is interpreted in terms of a structure in which helices C and E are coaxially stacked, with an acute angle subtended between helices C and D.

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 5′-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 concentration in 90 mM Tris-borate (pH 8.3), and plotted for the three vectors CD (■), DE (○), and CE (●). 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 junction e strand to its complement, to provide a model for coaxially stacked C and E arms. The different species were encapsulated in phospholipid 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 distributions. Histograms are shown for the CD junction, the CE junction, and the CE duplex. The measured mean E_FRET and half-widths for each distribution are presented in Table 1.

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 E_FRET for 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.

FIGURE 7.

Analysis of the structural flexibility of nucleotides in the IRES three-way junction using in-line probing. Radioactively [5′-³²P]-labeled RNA was incubated in 50 mM Tris-HCl (pH 8.3), 100 mM KCl, and 20 mM MgCl₂ for 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 5′-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).

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 5′-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.