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.

Visualizing large RNA molecules in solution

“…Helices at a 2-way junction can therefore remain collinear even though no significant electrostatic repulsions exist between them. Moreover, at a 3-helix junction, the collinearity of two helices due to coaxial stacking implicitly means that a plane is formed with the third (Kim et al 2002;Mikulecky et al 2004;Mohanty et al 2005;Ouellet et al 2010). Some 4-helix junctions have also been shown to have relatively planar structures (Lilley 1998(Lilley , 2000.…”

Lights, camera, action! Capturing the spliceosome and pre‐mRNA splicing with single‐molecule fluorescence microscopy

“…Both Hardin et al and Cornilescu et al used smFRET to study dynamics of a U4/U6 model di‐RNA . Both groups showed that the 3‐helical junction formed by the RNA was remarkably static with little evidence for dynamics on the millisecond to minute timescale, in agreement with studies of other 3‐helical junction RNAs . Hardin et al went on to propose that while the U4/U6 junction is static, the relative orientation of the U4 5′ stem loop can change upon addition of various U4/U6 di‐snRNP proteins with Prp31 promoting approach of the 5′ stem loop toward U4/U6 stem II and Prp3 and Prp4 promoting an increase in 5′ stem loop/stem II distance (Figure (c)).…”

“…56,78 Both groups showed that the 3-helical junction formed by the RNA was remarkably static with little evidence for dynamics on the millisecond to minute timescale, in agreement with studies of other 3-helical junction RNAs. 79 Hardin et al went on to propose that while the U4/U6 junction is static, the relative orientation of the U4 5 0 stem loop can change upon addition of various U4/U6 di-snRNP proteins with Prp31 promoting approach of the 5 0 stem loop toward U4/U6 stem II and Prp3 and Prp4 promoting an increase in 5 0 stem loop/stem II distance (Figure 3(c)). The exact correlation of smFRET data with U4/U6 structure is unclear: Hardin et al hypothesized that their smFRET data are consistent with coaxial stacking of U4/U6 stem I and stem II along with movement of this 5 0 stem loop.…”

Measurement of the Change in Twist at a Helical Junction in RNA Using the Orientation Dependence of FRET

“…We previously studied the structure and folding of this junction ( Fig. 1 B) (19). Analysis of the global structure indicated that in the presence of Mg 2þ ions, this junction adopts a conformation based on the coaxial alignment of the C and E helices, with helix D oriented to generate an acute angle with helix C (19).…”