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.

Institute of Biophysics Academy of Sciences of the Czech Republic Královopolská 135 61265 Brno Czech Republic

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Ban N, Nissen P, Hansen J, Moore PB, Steitz TA. The complete atomic structure of the large ribosomal subunit at 2.4 A resolution. Science. 2000;289:905–920. PubMed

Harms JM, Schluenzen F, Zarivach R, Bashan A, Gat S, Agmon I, Bartels H, Franceschi F, Yonath A. High resolution structure of the large ribosomal subunit from a mesophilic eubacterium. Cell. 2001;107:679–688. PubMed

Wimberly BT, Brodersen DE, Clemons WMJ, Morgan-Warren RJ, Carter AP, Vonrhein C, Hartsch T, Ramakrishnan V. Structure of the 30S ribosomal subunit. Nature. 2000;407:327–339. PubMed

Leontis NB, Westhof E. Analysis of RNA motifs. Curr. Opin. Struct. Biol. 2003;13:300–308. PubMed

Lee JC, Gutell RR, Russell R. The UAA/GAN internal loop motif: A new RNA structural element that forms a cross-strand AAA stack and long-range tertiary interactions. J. Mol. Biol. 2006;360:978–988. PubMed

Leontis NB, Stombaugh J, Westhof E. The non-Watson-Crick base pairs and their associated isostericity matrices. Nucleic Acids Res. 2002;30:3497–3531. PubMed PMC

Sarver M, Zirbel CL, Stombaugh J, Mokdad A, Leontis NB. FR3D: finding local and composite recurrent structural motifs in RNA 3D structures. J. Math. Biol. 2008;56:215–252. PubMed PMC

Jaeger L, Verzemnieks EJ, Geary C. The UA_handle: a versatile submotif in stable RNA architectures. Nucleic Acids Res. 2009;37:215–230. PubMed PMC

Schuwirth BS, Borovinskaya MA, Hau CW, Zhang W, Vila-Sanjurjo A, Holton JM, Cate JH. Structures of the bacterial ribosome at 3.5 Å resolution. Science. 2005;310:827–834. PubMed

Kazantsev AV, Krivenko AA, Harrington DJ, Holbrook SR, Adams PD, Pace NR. Crystal structure of a bacterial ribonuclease P RNA. Proc. Natl. Acad. Sci. U. S. A. 2005;102:13392–13397. PubMed PMC

Nissen P, Ippolito JA, Ban N, Moore PB, Steitz TA. RNA tertiary interactions in the large ribosomal subunit: The A-minor motif. Proc. Natl. Acad. Sci. U. S. A. 2001;98:4899–4903. PubMed PMC

Guillier M, Allemand F, Raibaud S, Dardel F, Springer M, Chiaruttini C. Translational feedback regulation of the gene for L35 in Escherichia coli requires binding of ribosomal protein L20 to two sites in its leader mRNA: A possible case of ribosomal RNA-messenger RNA molecular mimicry. RNA. 2002;8:878–889. PubMed PMC

Shankar N, Kennedy SD, Chen G, Krugh TR, Turner DH. The NMR structure of an internal loop from 23S ribosomal RNA differs from its structure in crystals of 50S ribosomal subunits. Biochemistry. 2006;45:11776–11789. PubMed PMC

Auffinger P, Bielecki L, Westhof E. Symmetric K+ and Mg2+ ion-binding sites in the 5S rRNA loop E inferred from molecular dynamics simulations. J. Mol. Biol. 2004;335:555–571. PubMed

Auffinger P, Hashem Y. Nucleic acid solvation: from outside to insight. Curr. Opin. Struct. Biol. 2007;17:325–333. PubMed

Cojocaru V, Klement R, Jovin TM. Loss of G–A base pairs is insufficient for achieving a large opening of U4 snRNA K-turn motif. Nucleic Acids Res. 2005;33:3435–3446. PubMed PMC

Deng NJ, Cieplak P. Molecular dynamics and free energy study of the conformational equilibria in the UUUU RNA hairpin. J. Chem. Theory Comput. 2007;3:1435–1450. PubMed

Krasovska MV, Sefcikova J, Spackova N, Sponer J, Walter NG. Structural dynamics of precursor and product of the RNA enzyme from the hepatitis delta virus as revealed by molecular dynamics simulations. J. Mol. Biol. 2005;351:731–748. PubMed

Li W, Sengupta J, Rath BK, Frank J. Functional conformations of the L11-ribosomal RNA complex revealed by correlative analysis of cryo-EM and molecular dynamics simulations. RNA. 2006;12:1240–1253. PubMed PMC

Reblova K, Fadrna E, Sarzynska J, Kulinski T, Kulhanek P, Ennifar E, Koca J, Sponer J. Conformations of flanking bases in HIV-1 RNA DIS kissing complexes studied by molecular dynamics. Biophys. J. 2007;93:3932–3949. PubMed PMC

Romanowska J, Setny P, Trylska J. Molecular dynamics study of the ribosomal A-site. J. Phys. Chem. B. 2008;112:15227–15243. PubMed PMC

Sanbonmatsu KY, Joseph S, Tung CS. Simulating movement of tRNA into the ribosome during decoding. Proc. Natl. Acad. Sci. U. S. A. 2005;102:15854–15859. PubMed PMC

Vaiana AC, Westhof E, Auffinger P. A molecular dynamics simulation study of an aminoglycoside/A-site RNA complex: conformational and hydration patterns. Biochimie. 2006;88:1061–1073. PubMed

Kopitz H, Zivkovic A, Engels JW, Gohlke H. Determinants of the unexpected stability of RNA fluorobenzene self pairs. Chembiochem. 2008;9:2619–2622. PubMed

Mazier S, Genest D. Insight into the intrinsic flexibility of the SL1 stem-loop from genomic RNA of HIV-1 as probed by molecular dynamics simulation. Biopolymers. 2008;89:187–196. PubMed

Mokdad A, Krasovska MV, Sponer J, Leontis NB. Structural and evolutionary classification of G/U wobble basepairs in the ribosome. Nucleic Acids Res. 2006;34:1326–1341. PubMed PMC

Rhodes MM, Reblova K, Sponer J, Walter NG. Trapped water molecules are essential to structural dynamics and function of a ribozyme. Proc. Natl. Acad. Sci. U. S. A. 2006;103:13380–13385. PubMed PMC

Schneider C, Brandl M, Suhnel J. Molecular dynamics simulation reveals conformational switching of water-mediated uracil-cytosine base-pairs in an RNA duplex. J. Mol. Biol. 2001;305:659–667. PubMed

Villa A, Wöhnert J, Stock G. Molecular dynamics simulation study of the binding of purine bases to the aptamer domain of the guanine sensing riboswitch. Nucleic Acids Res. 2009;37:4774–4786. PubMed PMC

Zhuang Z, Jaeger L, Shea JE. Probing the structural hierarchy and energy landscape of an RNA T-loop hairpin. Nucleic Acids Res. 2007;35:6995–7002. PubMed PMC

Razga F, Koca J, Sponer J, Leontis NB. Hinge-like motions in RNA kink-turns: The role of the second A-minor motif and nominally unpaired bases. Biophys. J. 2005;88:3466–3485. PubMed PMC

Spackova N, Sponer J. Molecular dynamics simulations of sarcin-ricin rRNA motif. Nucleic Acids Res. 2006;34:697–708. PubMed PMC

Reblova K, Razga F, Li W, Gao H, Frank J, Šponer J. Dynamics of the Base of Ribosomal A-site Finger Revealed by Molecular Dynamics Simulations and Cryo-EM. Nucleic Acids Res. 2009 doi: 10.1093/nar/gkp1057. PubMed PMC

Curuksu J, Sponer J, Zacharias M. Elbow Flexibility of the kt38 RNA Kink-Turn Motif Investigated by Free-Energy Molecular Dynamics Simulations. Biophys. J. 2009;97:2004–2013. PubMed PMC

Razga F, Koca J, Mokdad A, Sponer J. Elastic properties of ribosomal RNA building blocks: molecular dynamics of the GTPase-associated center rRNA. Nucleic Acids Res. 2007;35:4007–4017. PubMed PMC

Reblova K, Lankas F, Razga F, Krasovska MV, Koca J, Sponer J. Structure, dynamics, and elasticity of free 16S rRNA helix 44 studied by molecular dynamics simulations. Biopolymers. 2006;82:504–520. PubMed

Cheatham TE, III, Young MA. Molecular dynamics simulation of nucleic acids: successes, limitation, and promise. Biopolymers. 2001;56:232–256. PubMed

McDowell SE, Spackova N, Sponer J, Walter NG. Molecular dynamics simulations of RNA: An in silico single molecule approach. Biopolymers. 2007;85:169–184. PubMed PMC

Orozco M, Perez A, Noy A, Luque FJ. Theoretical methods for the simulation of nucleic acids. Chem. Soc. Rev. 2003;32:350–364. PubMed

Sponer J, Lankas F. Challenges and Advances in Computational Chemistry and Physics: Computational Studies of RNA and DNA. Dordrecht, Netherlands: Springer; 2006.

MacKerell AD, Nilsson L. Molecular dynamics simulations of nucleic acid-protein complexes. Curr. Opin. Struct. Biol. 2008;18:194–199. PubMed PMC

Orozco M, Noy A, Perez A. Recent advances in the study of nucleic acid flexibility by molecular dynamics. Curr. Opin. Struct. Biol. 2008;18:185–193. PubMed

Wang J, Cieplak P, Kollman PA. How Well Does a Restrained Electrostatic Potential (RESP) Model Perform in Calculating Conformational Energies of Organic and Biological Molecules? J. Comput. Chem. 2000;21:1049–1074.

Perez A, Marchan I, Svozil D, Sponer J, Cheatham TE, III, Laughton CA, Orozco M. Refinement of the amber force field for nucleic acids. Improving the description of α/γ conformers. Biophys. J. 2007;92:3817–3829. PubMed PMC

Klein DJ, Moore PB, Steitz TA. The Roles of Ribosomal Proteins in the Structure, Assembly and Evolution of the Large Ribosomal Subunit. J. Mol. Biol. 2004;340:141–177. PubMed

Selmer M, Dunham CM, Murphy FV, Weixlbaumer A, Petry S, Kelley AC, Weir JR, Ramakrishnan V. Structure of the 70S ribosome complexed with mRNA and tRNA. Science. 2006;313:1935–1942. PubMed

Gao YG, Selmer M, Dunham CM, Weixlbaumer A, Kelley AC, Ramakrishnan V. The Structure of the Ribosome with Elongation Factor G Trapped in the Posttranslocational State. Science. 2009;326:694–699. PubMed PMC

Schmeing TM, Voorhees RM, Kelley AC, Gao YG, Murphy FV, Weir JR, Ramakrishnan V. The Crystal Structure of the Ribosome Bound to EF-Tu and Aminoacyl-tRNA. Science. 2009;326:688–694. PubMed PMC

Zirbel CL, Sponer JE, Sponer J, Stombaugh J, Leontis NB. Classification and energetics of the base-phosphate interactions in RNA. Nucleic Acids Res. 2009;37:4898–4918. PubMed PMC

Pearlman DA, Case DA, Caldwell JW, Ross WS, Cheatham TE, III, DeBolt S. AMBER, a package of computer programs for applying molecular mechanics, normal mode analysis, molecular dynamics and free energy calculations to simulate the structural and energetic properties of molecule. Comput. Phys. Commun. 1995;91:1–41.

Case DA, Darden TA, Cheatham TE, III, Simmerling CL, Wang J, Duke RE, Luo R, Merz KM, Pearlman DA, Crowley M, Walker RC, Zhang W, Wang B, Hayik S, Roitberg A, Seabra G, Wong KF, Paesani F, Wu X, Brozell S, Tsui V, Gohlke H, Yang L, Tan C, Mongan J, Hornak V, Cui G, Beroza P, Mathews DH, Schafmeister C, Ross WS, Kollman PA. AMBER 9. San Francisco: University of California; 2006.

Cornell WD, Cieplak P, Bayly CI, Gould IR, Merz KM, Ferguson DM, Spellmeyer DC, Fox T, Caldwell JW, Kollman PA. A 2nd Generation Force-Field for the Simulation of Proteins, Nucleic-Acids, and Organic-Molecules. J. Am. Chem. Soc. 1995;117:5179–5197.

Aqvist J. Ion water interaction potentials derived from free-energy perturbation simulations. J. Phys. Chem. 1990;94:8021–8024.

Joung IS, Cheatham TE. Determination of alkali and halide monovalent ion parameters for use in explicitly solvated biomolecular simulations. J. Phys. Chem. B. 2008;112:9020–9041. PubMed PMC

Dang LX, Kollman PA. Free energy of association of the K+:18-crown-6 complex in water: a new molecular dynamics study. J. Phys. Chem. 1995;99:55–58.

Smith DE, Dang LX. Computer simulations of NaCl association in polarizable water. J. Chem. Phys. 1994;100:3757–3766.

Auffinger P, Cheatham TE, Vaiana AC. Spontaneous formation of KCl aggregates in biomolecular simulations: A force field issue? J. Chem. Theory Comput. 2007;3:1851–1859. PubMed

Berendsen HJC, Grigera JR, Straatsma TP. The missing term in effective pair potentials. J. Phys. Chem. 1987;91:6269–6271.

Humphrey W, Dalke A, Schulten K. VMD - Visual Molecular Dynamics. J. Mol. Graph. Model. 1996;14:33–38. PubMed

Zhou R, Berne BJ, Germain R. The free energy landscape for beta hairpin folding in explicit water. Proc. Natl. Acad. Sci. USA. 2001;18:14931–14936. PubMed PMC

Csaszar K, Spackova N, Stefl R, Sponer J, Leontis NB. Molecular dynamics of the frame-shifting pseudoknot from beet western yellows virus: The role of non- Watson-Crick base-pairing, ordered hydration, cation binding and base mutations on stability and unfolding. J. Mol. Biol. 2001;313:1073–1091. PubMed

Darden T, Pearlman D, Pedersen L. Ionic charging free energies: spherical versus periodic boundary condition. J. Phys. Chem. 1998;109:10921–10935.

Elber R, Karplus M. Enhanced sampling in molecular dynamics: use of the time dependent hartree approximation for a simulation of carbon monoxide diffusion through myoglobin. J. Am. Chem. Soc. 1990;112:9161–9175.

Roitberg A, Elber R. Modeling side chains in peptide and proteins: application of the locally enhanced sampling and the simulated annealing methods to find minimum energy conformations. J. Chem. Phys. 1991;95:9277–9287.

Simmerling C, Elber R. Hydrophobic "collapse" in a cyclic hexapeptide: Computer simulations of CHDLFC and CAAAAC in water. J. Am. Chem. Soc. 1994;116:2534–2547.

Fadrna E, Spackova N, Stefl R, Koca J, Cheatham TE, Sponer J. Molecular dynamics simulations of guanine quadruplex loops: Advances and force field limitations. Biophys. J. 2004;87:227–242. PubMed PMC

Simmerling C, Miller JL, Kollman PA. Combined locally enhanced sampling and Particle Mesh Ewald as a strategy to locate the experimental structure of a nonhelical nucleic acid. J. Am. Chem. Soc. 1998;120:7149–7155.

Fadrna E, Spackova N, Sarzynska J, Koca J, Orozco M, Cheatham TE, III, Kulinski T, Sponer J. Single stranded loops of quadruplex DNA as key benchmark for testing nucleic acids force fields. J. Chem. Theor. Comput. 2009;5:2514–2530. PubMed

Kollman PA, Massova I, Reyes C, Kuhn B, Huo SH, Chong L, Lee M, Lee T, Duan Y, Wang W, Donini O, Cieplak P, Srinivasan J, Case DA, Cheatham TE. Calculating structures and free energies of complex molecules: Combining molecular mechanics and continuum models. Accounts Chem. Res. 2000;33:889–897. PubMed

Srinivasan J, Cheatham TE, Cieplak P, Kollman PA, Case DA. Continuum solvent studies of the stability of DNA, RNA, and phosphoramidate - DNA helices. J. Am. Chem. Soc. 1998;120:9401–9409.

Luo R, David L, Gilson MK. Accelerated Poisson-Boltzmann calculations for static and dynamic systems. J. Comput. Chem. 2002;23:1244–1253. PubMed

Sitkoff D, Sharp KA, Honig B. Accurate calculation of hydration free energies using macroscopic solvent models. J. Phys. Chem. 1994;98:1978–1988.

Brooks B, Karplus M. Harmonic dynamics of proteins: Normal modes and fluctuations in bovine pancreatic trypsin inhibitor. Proc. Natl. Acad. Sci. U.S.A. 1983;80:6571–6575. PubMed PMC

Case DA, Darden TA, Cheatham TE, III, Simmerling CL, Wang J, Duke RE, Luo R, Crowley M, Ross WS, Zhang W, Merz KM, Wang B, Hayik S, Roitberg A, Seabra G, Kolossváry I, Wong KF, Paesani F, Vanicek J, Wu X, Brozell SR, Steinbrecher T, Gohlke H, Yang L, Tan C, Mongan J, Hornak V, Cui G, Mathews DH, Seetin MG, Sagui C, Babin V, Kollman PA. AMBER 10. San Francisco: University of California; 2008.

Alfonso DR, Jordan KD. A flexible nudged elastic band program for optimization of minimum energy pathways using ab initio electronic structure methods. J. Comput. Chem. 2003;24:990–996. PubMed

Jonsson H, Mills G, Jacobsen KW. Nudged elastic band method for finding minimum energy paths of transitions. In: Berne BJ, Ciccoti G, Coker DF, editors. Classical and Quantum Dynamics in Condensed Phase Simulations. Singapore: World Scientific; 1998. pp. 384–404.

Henkelman G, Jonsson H. Improved tangent estimate in the nudged elastic band method for finding minimum energy paths and saddle points. J. Chem. Phys. 2000;113:9978–9985.

Mathews DH, Case DA. Nudged elastic band calculation of minimal energy paths for the conformational change of a GG non-canonical pair. J. Mol. Biol. 2006;357:1683–1693. PubMed

Kumar S, Bouzida D, Swendsen RH, Kollman PA, Rosenberg JM. The weighted histogram analysis method for free-energy calculations on biomolecules. 1. The method. J. Comput. Chem. 1992;13:1011–1021.

Noy A, Pérez A, Laughton CA, Orozco M. Theoretical study of large conformational transitions in DNA: the B<->A conformational change in water and ethanol/water. Nucleic Acids Res. 2007;35:3330–3338. PubMed PMC

Aci S, Mazier S, Genest D. Conformational pathway for the kissing complex - Extended dimer transition of the SL1 stem-loop from genomic HIV-1 RNA as monitored by targeted molecular dynamics techniques. J. Mol. Biol. 2005;351:520–530. PubMed

Reblova K, Spackova N, Stefl R, Csaszar K, Koca J, Leontis NB, Sponer J. Non-Watson-Crick basepairing and hydration in RNA motifs: Molecular dynamics of 5S rRNA loop E. Biophys. J. 2003;84:3564–3582. PubMed PMC

Hobza P, Kabelac M, Sponer J, Mejzlik P, Vondrasek J. Performance of empirical potentials (AMBER, CFF95, CVFF, CHARMM, OPLS, POLTEV), semiempirical quantum chemical methods (AM1, MNDO/M, PM3), and ab initio Hartree-Fock method for interaction of DNA bases: Comparison with nonempirical beyond Hartree-Fock results. J. Comput. Chem. 1997;18:1136–1150.

Sponer JE, Spackova N, Kulhanek P, Leszczynski J, Sponer J. Non-Watson-Crick base pairing in RNA. Quantum chemical analysis of the cis Watson-Crick/sugar edge base pair family. J. Phys. Chem. A. 2005;109:2292–2301. PubMed

Vaiana AC, Sanbonmatsu KY. Stochastic Gating and Drug–Ribosome Interactions. J. Mol. Biol. 2009;386:648–661. PubMed PMC

Wood RH, Muhlbauer WCF, Thompson PT. Systematic errors in free energy perturbation calculations due to a finite sample of configuration space: sample size hysteresis. J. Phys. Chem. 1991;95:6670–6675.

Jucker FM, Heus HA, Yip PF, Moors EHM, Pardi A. A network of heterogeneous hydrogen bonds in GNRA tetraloops. J. Mol. Biol. 1996;264:968–980. PubMed

Mathews DH, Disney MD, Childs JL, Schroeder SJ, Zuker M, Turner DH. Incorporating chemical modification constraints into a dynamic programming algorithm for prediction of RNA secondary structure. Proc. Natl. Acad. Sci.U.S.A. 2004;101:7287–7292. PubMed PMC

Mathews DH, Sabina J, Zuker M, Turner DH. Expanded sequence dependence of thermodynamic parameters improves prediction of RNA secondary structure. J. Mol. Biol. 1999;288:911–940. PubMed

Xia T, SantaLucia J, Jr, Burkard ME, Kierzek R, Schroeder SJ, Jiao X, Cox C, Turner DH. Thermodynamic parameters for an expanded nearest-neighbor model for formation of RNA duplexes with Watson-Crick base pairs. Biochemistry. 1998;37:14719–14735. PubMed

Nowotny V, Nierhaus KH. Protein L20 from the large subunit of Escherichia coli ribosomes is an assembly protein. J. Mol. Biol. 1980;137:391–397. PubMed

Schneider B, Moravek Z, Berman HM. RNA conformational classes. Nucleic Acids Res. 2004;32:1666–1677. PubMed PMC

Besseova I, Otyeopka M, Reblova K, Sponer J. Dependence of A-RNA simulations on the choice of the force field and salt strength. Phys. Chem. Chem. Phys. 2009;11:10701–10711. PubMed

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