Conformations of an RNA Helix-Junction-Helix Construct Revealed by SAXS Refinement of MD Simulations

Abstract

RNA is involved in a broad range of biological processes that extend far beyond translation. Many of RNA's recently discovered functions rely on folding to a specific conformation or transitioning between conformations. The RNA structure contains rigid, short basepaired regions connected by more flexible linkers. Studies of model constructs such as small helix-junction-helix (HJH) motifs are useful in understanding how these elements work together to determine RNA conformation. Here, we reveal the full ensemble of solution structures assumed by a model RNA HJH. We apply small-angle x-ray scattering and an ensemble optimization method to selectively refine models generated by all-atom molecular dynamics simulations. The expectation of a broad distribution of helix orientations, at and above physiological ionic strength, is not met. Instead, this analysis shows that the HJH structures are dominated by two distinct conformations at moderate to high ionic strength. Atomic structures, selected from the molecular dynamics simulations, reveal strong base-base interactions in the junction that critically constrain the conformational space available to the HJH molecule and lead to a surprising re-extension at high salt. These results are corroborated by comparison with previous single-molecule fluorescence resonance energy transfer experiments on the same constructs.

Figure 1

HJH RNA construct and its sequence. The HJH molecule comprises three basic RNA elements: two 12-bp-long duplexes shown in dark and light colors, linked by an rU5 junction. The junction vector is defined by a bending angle θ between the z axis and the line joining the phosphorus atoms of C12 and C18. To see this figure in color, go online.

Figure 2

Comparison of raw SAXS data with predictions from the full (600-ns) MD pool and EOM refinements of that pool. (a) The raw SAXS profiles, the Kratky plot, and the pair distance distribution function. The Kratky plot of high salt suggests that HJH in high (500-mM) [KCl] is more extended than in low (30-mM) and medium (100-mM) [KCl]. The right two panels show the comparison with SAXS data of (b) MD and (c) EOM predictions, using residuals and χ² to evaluate the quality of the fits. The MD method fits the data best at medium salt and is improved, in all cases, by experimental refinement using EOM.

Figure 3

Distribution of radii of gyration (R_g values) of all structures at different [KCl] using Subpool-200 ns (a,d,g,j,m), Subpool-600 ns (b,e,h,k,n) and All-salt pool (c,f,i,l,o). The black dashed lines indicate the experimental R_g values of 27.5, 26.5, 24.7, 29.0, and 31.4 Å for 30–500 mM KCl concentrations. The gray histograms show the R_g distribution of all structures in each pool, under the quoted condition, whereas the darker and lighter histograms show the distributions from the “all-cycle” and “best-cycle” EOM analysis, respectively. Each distribution is normalized by the maximal number of counts. To see this figure in color, go online.

Figure 4

Number of base-stacking pairs in the junction (C12–18) within an ensemble at different [KCl]. Below [KCl] = 100 mM, the number of base-stacked pairs is small and mostly results from immobilization of C12 and (therefore) the highly constrained positions of U13. As [KCl] increases to 200 mM and beyond, the number increases significantly: two and three pairs are seen in 200 mM, whereas most of the bases are stacked along the junction at 500 mM. To see this figure in color, go online.

Figure 5

The calculated E_FRET of structures from the EOM-selected “all-cycle” ensemble as well as the full MD pool are plotted with the experimental data smFRET data (22). The error bars are the standard deviations of at leaset two independent measurements. The EOM-selected ensembles more closely capture the smFRET measurements. To see this figure in color, go online.

Figure 6

The spherical densities of the junction bending and twisting angles for structures in the optimized ensembles, shown as a function of [KCl]. The density of structures is indicated using the scale at right, with higher density regions indicated by a brighter “hot spot.” Motion of the hot spot is clearly seen, underscoring the observed structural transitions. Below ∼200 mM KCl, the angles increase. At high salt, an unexpected hot spot appears at small angles, reflecting extended states and stacked bases along the junction. To see this figure in color, go online.

Figure 7

Representative conformations of HJH in different [KCl] regimes: low (30 mM), medium (100 mM), and high (500 mM). The dominant conformation is shown in red, whereas other competing conformations are shown in transparent cyan, based on the detected frequencies in the ensemble. The junction conformations are shown on the right (C12-UUUUU-C18), with a label reflecting the percentage observed. This figure was generated by PyMOL (Schrödinger LLC, New York, NY) using home-written Python scripts. To see this figure in color, go online.