title: 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 (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.
authors.affiliation: Institute of Biophysics, Academy of Sciences of the Czech Republic, Královopolská 135, 61265 Brno, Czech Republic, National Centre for Biomolecular Research, Faculty of Science, Masaryk University, Kamenice 5, 62500 Brno, Czech Republic, Department of Biochemistry & Biophysics, University of Rochester Medical Center, 601 Elmwood Avenue, Box 712, Rochester, New York 14642, Department of Physics and Astronomy, University of Rochester, Rochester, New York 14627, and Department of Chemistry, University of Rochester, Rochester, New York 14627-0216.
authors.forename: Kamila
authors.initials: K
authors.lastname: Réblová
authors.affiliation: Institute of Biophysics, Academy of Sciences of the Czech Republic, Královopolská 135, 61265 Brno, Czech Republic, National Centre for Biomolecular Research, Faculty of Science, Masaryk University, Kamenice 5, 62500 Brno, Czech Republic, Department of Biochemistry & Biophysics, University of Rochester Medical Center, 601 Elmwood Avenue, Box 712, Rochester, New York 14642, Department of Physics and Astronomy, University of Rochester, Rochester, New York 14627, and Department of Chemistry, University of Rochester, Rochester, New York 14627-0216.
authors.forename: Zora
authors.initials: Z
authors.lastname: Střelcová
authors.affiliation: Institute of Biophysics, Academy of Sciences of the Czech Republic, Královopolská 135, 61265 Brno, Czech Republic, National Centre for Biomolecular Research, Faculty of Science, Masaryk University, Kamenice 5, 62500 Brno, Czech Republic, Department of Biochemistry & Biophysics, University of Rochester Medical Center, 601 Elmwood Avenue, Box 712, Rochester, New York 14642, Department of Physics and Astronomy, University of Rochester, Rochester, New York 14627, and Department of Chemistry, University of Rochester, Rochester, New York 14627-0216.
authors.forename: Petr
authors.initials: P
authors.lastname: Kulhánek
authors.affiliation: Institute of Biophysics, Academy of Sciences of the Czech Republic, Královopolská 135, 61265 Brno, Czech Republic, National Centre for Biomolecular Research, Faculty of Science, Masaryk University, Kamenice 5, 62500 Brno, Czech Republic, Department of Biochemistry & Biophysics, University of Rochester Medical Center, 601 Elmwood Avenue, Box 712, Rochester, New York 14642, Department of Physics and Astronomy, University of Rochester, Rochester, New York 14627, and Department of Chemistry, University of Rochester, Rochester, New York 14627-0216.
authors.forename: Ivana
authors.initials: I
authors.lastname: Beššeová
authors.affiliation: Institute of Biophysics, Academy of Sciences of the Czech Republic, Královopolská 135, 61265 Brno, Czech Republic, National Centre for Biomolecular Research, Faculty of Science, Masaryk University, Kamenice 5, 62500 Brno, Czech Republic, Department of Biochemistry & Biophysics, University of Rochester Medical Center, 601 Elmwood Avenue, Box 712, Rochester, New York 14642, Department of Physics and Astronomy, University of Rochester, Rochester, New York 14627, and Department of Chemistry, University of Rochester, Rochester, New York 14627-0216.
authors.forename: David H
authors.initials: DH
authors.lastname: Mathews
authors.affiliation: Institute of Biophysics, Academy of Sciences of the Czech Republic, Královopolská 135, 61265 Brno, Czech Republic, National Centre for Biomolecular Research, Faculty of Science, Masaryk University, Kamenice 5, 62500 Brno, Czech Republic, Department of Biochemistry & Biophysics, University of Rochester Medical Center, 601 Elmwood Avenue, Box 712, Rochester, New York 14642, Department of Physics and Astronomy, University of Rochester, Rochester, New York 14627, and Department of Chemistry, University of Rochester, Rochester, New York 14627-0216.
authors.forename: Keith
authors.initials: K
authors.lastname: Van Nostrand
authors.affiliation: Institute of Biophysics, Academy of Sciences of the Czech Republic, Královopolská 135, 61265 Brno, Czech Republic, National Centre for Biomolecular Research, Faculty of Science, Masaryk University, Kamenice 5, 62500 Brno, Czech Republic, Department of Biochemistry & Biophysics, University of Rochester Medical Center, 601 Elmwood Avenue, Box 712, Rochester, New York 14642, Department of Physics and Astronomy, University of Rochester, Rochester, New York 14627, and Department of Chemistry, University of Rochester, Rochester, New York 14627-0216.
authors.forename: Ilyas
authors.initials: I
authors.lastname: Yildirim
authors.affiliation: Institute of Biophysics, Academy of Sciences of the Czech Republic, Královopolská 135, 61265 Brno, Czech Republic, National Centre for Biomolecular Research, Faculty of Science, Masaryk University, Kamenice 5, 62500 Brno, Czech Republic, Department of Biochemistry & Biophysics, University of Rochester Medical Center, 601 Elmwood Avenue, Box 712, Rochester, New York 14642, Department of Physics and Astronomy, University of Rochester, Rochester, New York 14627, and Department of Chemistry, University of Rochester, Rochester, New York 14627-0216.
authors.forename: Douglas H
authors.initials: DH
authors.lastname: Turner
authors.affiliation: Institute of Biophysics, Academy of Sciences of the Czech Republic, Královopolská 135, 61265 Brno, Czech Republic, National Centre for Biomolecular Research, Faculty of Science, Masaryk University, Kamenice 5, 62500 Brno, Czech Republic, Department of Biochemistry & Biophysics, University of Rochester Medical Center, 601 Elmwood Avenue, Box 712, Rochester, New York 14642, Department of Physics and Astronomy, University of Rochester, Rochester, New York 14627, and Department of Chemistry, University of Rochester, Rochester, New York 14627-0216.
authors.forename: Jiří
authors.initials: J
authors.lastname: Šponer