An RNA molecular switch: Intrinsic flexibility of 23S rRNA Helices 40 and 68 5'-UAA/5'-GAN internal loops studied by molecular dynamics methods

Abstract

Functional RNA molecules such as ribosomal RNAs frequently contain highly conserved internal loops with a 5'-UAA/5'-GAN (UAA/GAN) consensus sequence. The UAA/GAN internal loops adopt distinctive structure inconsistent with secondary structure predictions. The structure has a narrow major groove and forms a trans Hoogsteen/Sugar edge (tHS) A/G base pair followed by an unpaired stacked adenine, a trans Watson-Crick/Hoogsteen (tWH) U/A base pair and finally by a bulged nucleotide (N). The structure is further stabilized by a three-adenine stack and base-phosphate interaction. In the ribosome, the UAA/GAN internal loops are involved in extensive tertiary contacts, mainly as donors of A-minor interactions. Further, this sequence can adopt an alternative 2D/3D pattern stabilized by a four-adenine stack involved in a smaller number of tertiary interactions. The solution structure of an isolated UAA/GAA internal loop shows substantially rearranged base pairing with three consecutive non-Watson-Crick base pairs. Its A/U base pair adopts an incomplete cis Watson-Crick/Sugar edge (cWS) A/U conformation instead of the expected Watson-Crick arrangement. We performed 3.1 µs of explicit solvent molecular dynamics (MD) simulations of the X-ray and NMR UAA/GAN structures, supplemented by MM-PBSA free energy calculations, locally enhanced sampling (LES) runs, targeted MD (TMD) and nudged elastic band (NEB) analysis. We compared parm99 and parmbsc0 force fields and net-neutralizing Na(+) vs. excess salt KCl ion environments. Both force fields provide a similar description of the simulated structures, with the parmbsc0 leading to modest narrowing of the major groove. The excess salt simulations also cause a similar effect. While the NMR structure is entirely stable in simulations, the simulated X-ray structure shows considerable widening of the major groove, loss of base-phosphate interaction and other instabilities. The alternative X-ray geometry even undergoes conformational transition towards the solution 2D structure. Free energy calculations confirm that the X-ray arrangement is less stable than the solution structure. LES, TMD and NEB provide a rather consistent pathway for interconversion between the X-ray and NMR structures. In simulations, the incomplete cWS A/U base pair of the NMR structure is water mediated and alternates with the canonical A-U base pair, which is not indicated by the NMR data. Completion of full cWS A/U base pair is prevented by the overall internal loop arrangement. In summary, the simulations confirm that the UAA/GAN internal loop is a molecular switch RNA module that adopts its functional geometry upon specific tertiary contexts.

Figure 1

2D structures (Left) and 3D stereo views (Right) of studied segments including non-Watson-Cricks base pairs in the internal UAA/GAA loop (Middle). A. Left: 2D X-ray structure of H40 with unified sequence flanking the internal loop (see Materials and Methods). Middle: sheared A/G and rH U/A base pairs. Right: Stereo view of E.c. H40 X-ray structure. B. Left: 2D X-ray structure of E.c. 23S rRNA H68 exhibiting an alternative conformation of UAA/GAA motif with unified canonical flanking sequence. Black dashed line indicates single H-bond. Middle: unpaired G and A bases, stacking middle adenines and single bonded A/U base pair. Right: Stereo view of this structure. C. Left: 2D NMR structure. Middle: sheared A/G, sheared A/A and incomplete cWS A/U base pairs. Right: Stereo view of the NMR structure. In all Figures, bases of the UAA/GAA internal loop are in red, 3D structures are colored accordingly, hydrogens are not shown in the X-ray structures, bases in yellow boxes in the 2D structures are involved in stacking and the marks between the bases indicate base paring family according to the Leontis & Westhof classification (tHS = trans Hoogsteen/Sugar edge A/G or A/A, known also as “sheared” base pairs, tWH = trans Watson-Crick/Hoogsteen U/A, known also as reverse Hoogsteen (rH) base pair, and cWS = cis Watson-Crick/Sugar edge A/U)., X-ray nucleotide numbers are in blue, NMR numbers are in black. The green rectangular trapezium for H40 structure marks bases forming the UA_handle submotif.

Figure 2

Base phosphate interactions observed in the ribosomal X-ray structures of H40. E.c. structure exhibits bifurcated binding mode (base phosphate interaction type 4BPh) in which N2 and N1 of G13 bind to the same anionic oxygen of the phosphate group A5(O2P). H.m., D.r. and T.t. structures exhibit only G13(N1)-A5(O2P) H-bond, which represents base phosphate interaction type 5BPh. The differences might reflect limits of the resolution of the experimental structures.

Figure 3

A. Stereo view of three adenines of the UAA/GAA motif from E.c. H40 forming an AAA stack which interacts with two C=G base pairs from the hairpin between H39 and H40 via A-minor interactions. C=G pair and the corresponding interacting adenine(s) are highlighted with the same color. Details of these interactions are visualized below the stereo view in corresponding green and red boxes, including a description of the A-minor interaction type. B. Stereo view of bacterial E.c. H40 interacting with hairpin between H39 (in green) and H40 and ribosomal protein L20 (in magenta). C. Stereo view of archaeal H.m. H40 interacting with the hairpin between H39 (in green) and H40, ribosomal protein L30 (in yellow) and H25 (in grey). D. Stereo view of bacterial E.c. H68 (exhibiting the alternative conformation of UAA/GAN internal loop) interacting with H75 (in grey).

Figure 4

A. Stereo view of the averaged 55–57 ns MD structure of the simulated X-ray H40 UAA/GAA internal loop from the MD_Ec_99 simulation. The structure exhibits wider (more open) major groove compared to the original geometry (see Figure 1A). Monitored inter-phosphate distances across the major groove are indicated by black (12P-3P) and red arrows (11P-4P) in blue transparent boxes. B. Time courses of two inter-phosphate distances (12P-3P in black and 11P-4P in red) in standard MD simulations of X-ray H40 run with the parm99 force field and in control simulations run with the parmbsc0 force field, all with net-neutralizing Na⁺ (Table 1). The x-axis stands for time (in nanoseconds) while y-axis stands for inter-phosphate distance (in Å). Horizontal lines show experimental distances. C. Time course of two inter-phosphate distances (12P-3P in black and 11P-4P in red) in standard MD simulation of NMR structure run with the parm99 force field and in control simulation run with the parmbsc0 force field.

Figure 5

A. Time courses of the van der Waals interaction energy calculated between bases forming A5A6, A5A14 and A5G13 stacks in the standard simulations of X-ray H40 run with parm99 force field. B. Stereo views of X-ray H40 UAA/GAA internal loop with colored bases forming stacks. Top – the original stacking pattern highlighted by the black oval, i.e. A5 forms an intrastrand stack with A6, and simultaneously cross-strand stack with A14. Bottom – the stacking pattern formed in the course of the simulations (highlighted by the black oval) where A5 stacks with G13 and also with A14.

Figure 6

A. Stereo view of the snapshot structure of the H68 UAA/GAA internal loop from the MD_68 simulation at 5 ns. The structure exhibits a wider (more open) major groove compared to the original geometry (see Figure 1B) B. Time courses of two inter-phosphate distances (12P-3P in black and 11P-4P in red) along the MD_68 simulation. C. Stereo view of the snapshot structure of the H68 UAA/GAA internal loop from the MD_68 simulation at 40 ns with formed sheared A14/A5 and cWS A15/U4 pairs, which are highlighted in the color transparent boxes. This structure closely resembles the NMR structure.

Figure 7

3D stereo view of a snapshot of the UAA/GAA structure from the LES_Ec simulation with multiple copies of nucleotides in the internal loop (LES region). The structure resembles the solution structure (Figure 1C), i.e. it has wide major groove and coplanar A6 and G13 (red), A5 and A14 (blue) and U4 and A15 (green) bases.

Figure 8

Total free energy time courses in standard net-neutralizing Na⁺ simulations with parm99 force field. The x-axes stand for time (in nanoseconds) while y-axes stand for total free energy (in kcal/mol). The grey vertical dashed lines mark the time period when initial opening of the major groove was observed. In the MD_Tt_99 simulation the major groove oscillated back and forth with the grey lines indicating the first opening. In the time course of the MD_H68 simulation the vertical black dashed lines indicate the time period when the sheared A/A and cWS A/U pairs formed.

Figure 9

3D stereo view of structures showing the conformational transition between NMR and X-ray structures predicted by NEB calculations. G13 is highlighted in red, A6 in green, A14 in blue, A5 in yellow, A15 in magenta and U4 in cyan. A) Starting NMR structure. B) Intermediate structure where the A14/A5 pair breaks and moves away from being stacked with A6 and A15. C) Intermediate structure where A15 slides out of the helix to become bulged out. D) Final structure where A14 pairs with U4 while A5 is unpaired and stacks with A6. This structure corresponds to the arrangement of the H40 X-ray structure.