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.

Computational Studies of the Cholesterol Transport between NPC2 and the N-Terminal Domain of NPC1 (NPC1(NTD))

“…This method has been successfully applied to a number of biological processes of similar complexity. 51,52 We have used the NEB approach to model the pathway for cholesterol transfer between the NPC1(NTD) and NPC2 Figure 9, discussed in more detail in the text. The colors of the arrows match the colors of the cholesterol molecule in the images shown in Figure 9 for different steps of the transfer.…”

Structural Dynamics of the Box C/D RNA Kink-Turn and Its Complex with Proteins: The Role of the A-Minor 0 Interaction, Long-Residency Water Bridges, and Structural Ion-Binding Sites Revealed by Molecular Simulations

“…The parm99 and parmbsc0 force fields appear to provide equivalent (within the limits of the sampling achieved in our work) description of most parts of the present system, in agreement with other recent tests. 62,63 The parmbsc0 obviously penalizes the γ-trans backbone substates. This, however, usually does not have a substantial effect on the RNA behavior, and we cannot make any recommendation which of the two force fields is closer to reality.…”

HIV-1 TAR RNA Spontaneously Undergoes Relevant Apo-to-Holo Conformational Transitions in Molecular Dynamics and Constrained Geometrical Simulations

“…Moreover, the force field variants parm94, parm99, and parmbsc0 all resulted in overall similar descriptions of RNA structures in simulations of up to 100 ns length in previous studies. 54,55 Thus, we expect that the main results of our simulations will not be sensitive to the used Amber force field variant.…”

“…50,51 A recent reparameterization of the R and γ torsions in these force fields led to the parmbsc0 force field, 52 which improved the simulation of DNA duplexes on very long time scales (1 µs range). However, further tests on A-RNA duplexes 53 and UAA/GAN internal loops of 23S rRNA H40 54 revealed that the penalization of the R/γ transitions in the parmbsc0 force field may be too excessive for RNA structures and leads to a modest narrowing of the major groove due to the suppression of the γ-trans states. 54 In the present study, when investigating R and γ torsions sampled during MD and REMD simulations using parm94, we found only one to two transitions toward R/γ t/t substates, e.g., for nucleotides 20 and 28 in the two stem regions.…”

“…However, further tests on A-RNA duplexes 53 and UAA/GAN internal loops of 23S rRNA H40 54 revealed that the penalization of the R/γ transitions in the parmbsc0 force field may be too excessive for RNA structures and leads to a modest narrowing of the major groove due to the suppression of the γ-trans states. 54 In the present study, when investigating R and γ torsions sampled during MD and REMD simulations using parm94, we found only one to two transitions toward R/γ t/t substates, e.g., for nucleotides 20 and 28 in the two stem regions. This result agrees with the occasional finding of these substates in experimental structures 54 and, thus, supports the validity of our simulations.…”