Loss of G-A base pairs is insufficient for achieving a large opening of U4 snRNA K-turn motif

Abstract

Upon binding to the 15.5K protein, two tandem-sheared G-A base pairs are formed in the internal loop of the kink-turn motif of U4 snRNA (Kt-U4). We have reported that the folding of Kt-U4 is assisted by protein binding. Unstable interactions that contribute to a large opening of the free RNA ('k-e motion') were identified using locally enhanced sampling molecular dynamics simulations, results that agree with experiments. A detailed analysis of the simulations reveals that the k-e motion in Kt-U4 is triggered both by loss of G-A base pairs in the internal loop and backbone flexibility in the stems. Essential dynamics show that the loss of G-A base pairs is correlated along the first mode but anti-correlated along the third mode with the k-e motion. Moreover, when enhanced sampling was confined to the internal loop, the RNA adopted an alternative conformation characterized by a sharper kink, opening of G-A base pairs and modified stacking interactions. Thus, loss of G-A base pairs is insufficient for achieving a large opening of the free RNA. These findings, supported by previously published RNA structure probing experiments, suggest that G-A base pair formation occurs upon protein binding, thereby stabilizing a selective orientation of the stems.

Figure 1

Different conformations of Kt-U4; guanines are shown in blue, cytosines in orange, adenines in red and uracils in yellow; long arrows indicate newly formed stacking patters; the short arrows indicate the kink in the backbone; ϕ is the angle between the P atoms of C47, U31 and G35. (A) Kt-U4 RNA structure from the crystallographic structure of Kt-U4–15.5K complex with the UUUAU external loop modeled. (B) Open conformation observed during the LES4 trajectory. (C) Tightly kinked alternative conformation captured in the LES1 trajectory. (D) Structure of the Kt58 for comparison with the structure in Figure 1C.

Figure 2

Correlation between opening of G–A base pairs and the k–e motion. (A) The ϕ angle during the LES4 (black), LES1 (red) and LES2 (blue) trajectories. (B) The distance d between N2 of G32 and N7 of A44 during the LES4 (black), LES1 (red) and LES2 (blue) trajectories. (C) 2D diagram of d(ϕ) in the LES4 trajectory. (D) 2D diagram of d(ϕ) in the LES1 trajectory. In (C) and (D), the maximal donor–acceptor distance (3.2 Å) for hydrogen-bond interaction is indicated with a horizontal line.

Figure 3

Scatter 3D plots of the stacking interactions between: (A) G32/G43 in the LES4 trajectory; (B) G32/G43 in the LES1 trajectory; (C) A44/A33 in the LES4 trajectory; (D) A44/A33 in the LES1 trajectory. Stacking interactions occur when the distance (D) is minimal and the dihedral angle (θ) between the planes of the nucleotides is either ∼0° or ∼180° between the planes of the nucleotides. Rotation of the base about the N9–C1′ bond occurs when θ approaches intermediate values. The scatter points are colored according to their y value (D) with a colormap ranging from blue to red.

Figure 4

Scatter 3D plots of the stacking interactions between: (A) A44/G32 in the LES4 trajectory; (B) A44/G32 in the LES1 trajectory; (C) G43/A33 in the LES4 trajectory; (D) G43/A33 in the LES1 trajectory. For details clarifying the plots and coloring see Figure 3.

Figure 5

Structural view of the transitions between different stacking patterns in the free RNA. (A) G32/G43 and A44/A33 stacking interactions as observed in the crystal structure of Kt-U4–15.5K complex. (B) A44/G32/G43 stacking interactions formed in the LES4 trajectory after ∼7 ns. (C) Rotation about the N9–C1′ bond in A33 and A44 as observed in the LES4 trajectory after ∼9 ns. (D) G32/G43/A33 stacking interactions formed in the LES1 trajectory. Guanines are blue, adenines red, oxygens pink, nitrogens cyan and the sugar–phosphate backbone gray.

Figure 6

Correlation between backbone flexibility and the k–e motion. (A) The pseudorotation angle describing the sugar pucker of A33 during the LES4 (filled circles) and LES1 (empty circles) trajectories. (B) The χ angle describing the rotation about N9–C1′ bond in A33 during the LES4 (filled circles) and LES1 (empty circles) trajectories. (C) relative populations of ±ap (black) and +sc (gray) configurations (describing the rotation about the C4′–C5′ bond) in G35, G46 and G45 in the LES4, LES1 and LES2 trajectories; the percentage of trajectory frames, in which intermediate configurations were sampled is not shown.

Figure 7

PCA of LES4 trajectory. (A) Decomposition of the trajectory along the first 25 modes. (B) Projection of the LES4 trajectory onto first (continuous line) and third (dotted line with square symbols) modes. (C) ϕ angle along the first mode. (D) Distance between N2 of G43 and N7 of A33 along the first mode. (E) ϕ angle along the third mode. (F) Distance between N2 of G43 and N7 of A33 along the third mode.

Figure 8

Protein cavity at the protein–RNA interface; α-helical regions are shown in purple, β-sheet regions in yellow and unstructured regions in cyan. For RNA coloring see Figure 1.