Do conformational biases of simple helical junctions influence RNA folding stability and specificity?

Abstract

Structured RNAs must fold into their native structures and discriminate against a large number of alternative ones, an especially difficult task given the limited information content of RNA's nucleotide alphabet. The simplest motifs within structured RNAs are two helices joined by nonhelical junctions. To uncover the fundamental behavior of these motifs and to elucidate the underlying physical forces and challenges faced by structured RNAs, we computationally and experimentally studied a tethered duplex model system composed of two helices joined by flexible single- or double-stranded polyethylene glycol tethers, whose lengths correspond to those typically observed in junctions from structured RNAs. To dissect the thermodynamic properties of these simple motifs, we computationally probed how junction topology, electrostatics, and tertiary contact location influenced folding stability. Small-angle X-ray scattering was used to assess our predictions. Single- or double-stranded junctions, independent of sequence, greatly reduce the space of allowed helical conformations and influencing the preferred location and orientation of their adjoining helices. A double-stranded junction guides the helices along a hinge-like pathway. In contrast, a single-stranded junction samples a broader set of conformations and has different preferences than the double-stranded junction. In turn, these preferences determine the stability and distinct specificities of tertiary structure formation. These sequence-independent effects suggest that properties as simple as a junction's topology can generally define the accessible conformational space, thereby stabilizing desired structures and assisting in discriminating against misfolded structures. Thus, junction topology provides a fundamental strategy for transcending the limitations imposed by the low information content of RNA primary sequence.

FIGURE 1.

(A) Visualization of the dPEG (left) and sPEG (right) HJH constructs. Both constructs are composed of two 12 bp DNA duplexes (colored in gray) and double- or single-stranded PEG junctions of six ethylene–glycol monomers (green and red). The 5′ and 3′ oxygens proximal to the junction are exaggerated and highlighted in magenta and orange, respectively. (B) Angled view (left) and end-on view (right) of the standard orthonormal body frame attached to each helix. The axis was chosen to point along the helical axis, while the axis was chosen to point orthogonally relative to the axis in the direction of the 3′ oxygen of the terminal residue. The axis was perpendicular to the and axes and computed as . As noted, the polar angle φ subtended by the 5′ and 3′ oxygens is approximately −133°.

FIGURE 2.

Visualization of 1000 randomly selected conformers observed in the dPEG (A) and sPEG (B) simulations. One helix, designated as a “fixed” reference helix, is colored in gray, while the other “mobile” helix is depicted as a cylinder whose radius has been reduced for clarity. The junction atoms have been hidden in this view. The differences between the ensembles induced by the different junctions are obvious; while the dPEG junction largely constrains the helices to a plane, yielding a “Mohawk”-shaped ensemble, the sPEG junction allows the helices to move with greater freedom, resulting in a broad “umbrella”-shaped ensemble. The φ–φ scatter plots reveal the tendency of the dPEG (C) and sPEG (D) junctions to bring different faces of the helices into close proximity. Each black dot represents the φ–φ measure of an individual helical configuration observed during simulation whose interhelical angle is less than 45°. Contours have been added to highlight the density of the distribution and each contour represents a 10% decrease from the maximum density. The dPEG junction tends to bring the −85° face of the fixed helix together with the −85° face of the mobile helix, consistent with a hinge-like motion. Changing the dPEG into a sPEG junction alters this hinge-like motion, makes the distribution broader and brings together the −4° and the −143° faces of the fixed and mobile helices (see also Figs. 1B, 3).

FIGURE 3.

Schematic representation of the sPEG (top row) and dPEG (bottom row) HJH motifs in a side-by-side helical configuration. The left column is a view of the HJH motif down the helical axis and the right column is a view from the side. As in Fig. 1B, the 3′ and 5′ oxygens proximal to the junction are respectively, colored orange and magenta. To study folding specificity, tertiary contacts in three separate locations (indicated by ♦, •, and ▴ symbols) were computationally added to the ends of the helices on different helical faces (left column). In the standard body coordinate system, the diamond, circle, and triangle locations are, respectively, on the (0°,−133°), (−67°,−67°), and (−133°,0°) faces (shaded symbols, left panel). The topology of the sPEG and dPEG junctions introduces conformational biases that tend to promote the formation of different tertiary contacts; sPEG junctions promote formation of the diamond contact, while dPEG junctions promote the formation of the circle contact (solid symbols, right column).

FIGURE 4.

Comparison between predicted (—) and experimentally measured (○) SAXS profiles for dPEG (A) and sPEG (B) tethered duplex constructs over a range of Na⁺ conditions (plotted as scattering intensity I(s) weighted by s). Na⁺ concentrations (from bottom to top) were: 0.016, 0.056, 0.116, 0.416, 0.616, and 1.016 M, with the addition of an additional profile at 0.216 M for dPEG. For clarity, profiles were vertically offset and the number of data points reduced by one-third. Error bars are smaller than symbols for some points. The disappearance of the second peak (roughly located at s = 0.02 Å⁻¹) with increasing salt indicates that the tethered duplex is relaxing in response to the screening of the increased salt concentration.

FIGURE 5.

Simulated folding of sPEG (top row) and dPEG (bottom row) motifs. Salt-induced folding progress curves, in terms of fraction folded (P_F), for HJH motifs with three tertiary contact locations (♦, •, ▴) and sPEG (A) and dPEG (D) junctions. Corresponding values for ΔG_fold are plotted in (B) and (E). Values for ΔU_fold (- -) and TΔS_fold (—) are plotted in (C) and (F). ΔU_fold includes a constant contribution from the tertiary contact (−14.5k_BT for both junctions). The horizontal black dotted lines in C and F denote the average value of ΔS_fold over the range of salt conditions.

FIGURE 6.

Comparison of the fraction folded at 1 M monovalent salt concentration for the different junction topologies and tertiary contact locations. The two junction topologies have different conformational preferences (sPEG prefers the diamond position, dPEG prefers circle) and folding specificities (sPEG is more resilient to changes in tertiary contact location). In actual motifs, the observed specificity would be somewhat reduced because the helices could fray to facilitate tertiary contact formation in regions of conformational space that would otherwise be disallowed (see the text).