An RNA Molecular Switch: Intrinsic Flexibility of 23S rRNA Helices 40 and 68 5′-UAA/5′-GAN Internal Loops Studied by Molecular Dynamics Methods

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• 作者：Kamila Rblov ; Zora Stelcov ; Petr Kulhnek ; Ivana Beeov ; David H. Mathews ; Keith Van Nostrand ; Ilyas Yildirim ; Douglas H. Turner ; Ji poner
• 刊名：Journal of Chemical Theory and Computation
• 出版年：2010
• 出版时间：March 9, 2010
• 年：2010
• 卷：6
• 期：3
• 页码：910-929
• 全文大小：833K
• 年卷期：v.6,no.3(March 9, 2010)
• ISSN：1549-9626

文摘

Functional RNA molecules such as ribosomal RNAs (rRNAs) frequently contain highly conserved internal loops with a 5′-UAA/5′-GAN (UAA/GAN) consensus sequence. The UAA/GAN internal loops adopt a 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 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 molecular mechanics, Poisson−Boltzmann, and surface area 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⁺ versus 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, a loss of base−phosphate interaction, and other instabilities. The alternative X-ray geometry even undergoes a conformational transition toward 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 the 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.