Helix 38 (H38) of the large ribosomal subunit, with a length of 110 A, reaches the small subunit through intersubunit bridge B1a. Previous cryo-EM studies revealed that the tip of H38 moves by more than 10 A from the non-ratcheted to the ratcheted state of the ribosome while mutational studies implicated a key role of flexible H38 in attenuation of translocation and in dynamical signaling between ribosomal functional centers. We investigate a region including the elbow-shaped kink-turn (Kt-38) in the Haloarcula marismortui archaeal ribosome, and equivalently positioned elbows in three eubacterial species, located at the H38 base. We performed explicit solvent molecular dynamics simulations on the H38 elbows in all four species. They are formed by at first sight unrelated sequences resulting in diverse base interactions but built with the same overall topology, as shown by X-ray crystallography. The elbows display similar fluctuations and intrinsic flexibilities in simulations indicating that the eubacterial H38 elbows are structural and dynamical analogs of archaeal Kt-38. We suggest that this structural element plays a pivotal role in the large motions of H38 and may act as fulcrum for the abovementioned tip motion. The directional flexibility inferred from simulations correlates well with the cryo-EM results.

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

Zobrazit více v PubMed

Yusupov MM, Yusupova GZ, Baucom A, Lieberman K, Earnest TN, Cate JHD, Noller HF. Crystal structure of the ribosome at 5.5 angstrom resolution. Science. 2001;292:883–896. PubMed

Frank J, Agrawal RK. A ratchet-like inter-subunit reorganization of the ribosome during translocation. Nature. 2000;406:318–322. PubMed

Valle M, Zavialov A, Sengupta J, Rawat U, Ehrenberg M, Frank J. Locking and unlocking of ribosomal motions. Cell. 2003;114:123–134. PubMed

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. USA. 2001;98:4899–4903. PubMed PMC

Liiv A, O'C;onnor M. Mutations in the intersubunit bridge regions of 23 S rRNA. J. Biol. Chem. 2006;281:29850–29862. PubMed

Piekna-Przybylska D, Przybylski P, Baudin-Baillieu AS, Rousset JP, Fournier MJ. Ribosome performance is enhanced by a rich cluster of pseudouridines in the A-site finger region of the large subunit. J. Biol. Chem. 2008;283:26026–26036. PubMed PMC

Yassin A, Mankin AS. Potential new antibiotic sites in the ribosome revealed by deleterious mutations in RNA of the large ribosomal subunit. J. Biol. Chem. 2007;282:24329–24342. PubMed

Komoda T, Sato NS, Phelps SS, Namba N, Joseph S, Suzuki T. The a-site finger in 23 S rRNA acts as a functional attenuator for translocation. J. Biol. Chem. 2006;281:32303–32309. PubMed

Rakauskaite R, Dinman JD. An arc of unpaired ‘hinge bases’ facilitates information exchange among functional centers of the ribosome. Mol. Cell. Biol. 2006;26:8992–9002. PubMed PMC

Sergiev PV, Kiparisov SV, Burakovsky DE, Lesnyak DV, Leonov AA, Bogdanov AA, Dontsova OA. The conserved A-site finger of the 23 S rRNA: Just one of the intersubunit bridges or a part of the allosteric communication pathway? J. Mol. Biol. 2005;353:116–123. PubMed

Agirrezabala X, Lei JL, Brunelle JL, Ortiz-Meoz RF, Green R, Frank J. Visualization of the hybrid state of tRNA binding promoted by spontaneous ratcheting of the ribosome. Mol. Cell. 2008;32:190–197. PubMed PMC

Frank J, Gao HX, Sengupta J, Gao N, Taylor DJ. The process of mRNA-tRNA translocation. Proc. Natl Acad. Sci. USA. 2007;104:19671–19678. PubMed PMC

Gao HX, Zhou ZH, Rawat U, Huang C, Bouakaz L, Wang CH, Cheng ZH, Liu YY, Zavialov A, Gursky R, et al. RF3 induces ribosomal conformational changes responsible for dissociation of class I release factors. Cell. 2007;129:929–941. PubMed

Valle M, Sengupta J, Swami NK, Grassucci RA, Burkhardt N, Nierhaus KH, Agrawal RK, Frank J. Cryo-EM reveals an active role for aminoacyl-tRNA in the accommodation process. EMBO J. 2002;21:3557–3567. 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

Lescoute A, Leontis NB, Massire C, Westhof E. Recurrent structural RNA motifs, isostericity matrices and sequence alignments. Nucleic Acids Res. 2005;33:2395–2409. PubMed PMC

Klein DJ, Schmeing TM, Moore PB, Steitz TA. The kink-turn: a new RNA secondary structure motif. EMBO J. 2001;20:4214–4221. PubMed PMC

Razga F, Spackova N, Reblova K, Koca J, Leontis NB, Sponer J. Ribosomal RNA kink-turn motif - A flexible molecular hinge. J. Biomol. Struct. Dyn. 2004;22:183–193. PubMed

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

Razga F, Zacharias M, Reblova K, Koca J, Sponer J. RNA kink-turns as molecular elbows: Hydration, cation binding, and large-scale dynamics. Structure. 2006;14:825–835. PubMed

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

Curuksu J, Šponer J, Zacharias M. The elbow flexibility of the kt38 RNA kink turn motif investigated by free energy molecular dynamics simulations. Biophys. J. 2009;97:2004–2013. 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 A resolution. Science. 2005;310:827–834. 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

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

Korostelev A, Trakhanov S, Asahara H, Laurberg M, Lancaster L, Noller HF. Interactions and dynamics of the Shine Dalgarno helix in the 70S ribosome. Proc. Natl Acad. Sci. USA. 2007;104:16840–16843. PubMed PMC

Blanchard SC, Gonzalez RL, Kim HD, Chu S, Puglisi JD. tRNA selection and kinetic proofreading in translation. Nat. Struct. Mol. Biol. 2004;11:1008–1014. PubMed

Blanchard SC, Kim HD, Gonzalez RL, Puglisi JD, Chu S. tRNA dynamics on the ribosome during translation. Proc. Natl Acad. Sci. USA. 2004;101:12893–12898. PubMed PMC

Dorywalska M, Blanchard SC, Gonzalez RL, Kim HD, Chu S, Puglisi JD. Site-specific labeling of the ribosome for single-molecule spectroscopy. Nucleic Acids Res. 2005;33:182–189. PubMed PMC

Ninio J. Multiple stages in codon-anticodon recognition: double-trigger mechanisms and geometric constraints. Biochimie. 2006;88:963–992. PubMed

Spirin AS. Ribosome as a molecular machine. FEBS Lett. 2002;514:2–10. 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

Auffinger P, Bielecki L, Westhof E. Symmetric K+ and Mg2+ ion-binding sites in the 5 S 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

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

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

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

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

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

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

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

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

Pearlman DA, Case DA, Caldwell JW, Ross WS, Cheatham T.E., 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 T.E., III, Simmerling CL, Wang J, Duke RE, Luo R, Merz KM, Wang B, Pearlman DA, et al. AMBER 8. San Francisco: University of California; 2004.

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.

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

Darden T, York D, Pedersen L. Particle Mesh Ewald-an N.Log(N) method for Ewald sums in large systems. J. Chem. Phys. 1993;98:10089–10092.

Humphrey W, Dalke A, Schulten K. VMD – visual molecular dynamics. J. Mol. Graph. Model. 1996;14:33–38. PubMed

DeLano WL. DeLano Scientific. CA, USA: Palo Alto; 2002. The PyMOL Molecular Graphics System.

Lindahl E, Hess B, van der Spoel V. GROMACS 3.0: a package for molecular simulation and trajectory analysis. J. Mol. Model. 2001;7:306–317.

Amadei A, Linssen ABM, Berendsen HJC. Essential dynamics of proteins. Proteins. 1993;17:412–425. PubMed

Mongan J. Interactive essential dynamics. J. Comput. Aid. Mol. Des. 2004;18:433–436. PubMed

Shao JY, Tanner SW, Thompson N, Cheatham TE. Clustering molecular dynamics trajectories: 1. Characterizing the performance of different clustering algorithms. J. Chem. Theory Comput. 2007;3:2312–2334. PubMed

Feig M, Karanicolas J, Brooks CL. MMTSB Tool Set: enhanced sampling and multiscale modeling methods for applications in structural biology. J. Mol. Graph. Model. 2004;22:377–395. PubMed

Case DA, Darden TA, Cheatham T.E., III, Simmerling CL, Wang J, Duke RE, Luo R, Crowley M, Ross WS, Zhang W, et al. AMBER 10. San Francisco: University of California; 2008.

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

Hashem Y, Auffinger P. A short guide for molecular dynamics simulations of RNA systems. Methods. 2009;47:187–197. PubMed

Valle M, Zavialov A, Li W, Stagg SM, Sengupta J, Nielsen RC, Nissen P, Harvey SC, Ehrenberg M, Frank J. Incorporation of aminoacyl-tRNA into the ribosome as seen by cryo-electron microscopy. Nat. Struct. Biol. 2003;10:899–906. PubMed

Chapman MS. Restrained real-space macromolecular atomic refinement using a new resolution-dependent electron-density function. Acta Cryst. A. 1995;51:69–80.

Gao H, LeBarron J, Frank J. Ribosomal dynamics – intrinsic instability of a molecular machine. In: Walter NG, Woodson SA, Batey RT, editors. Non-protein Coding RNAs. Berlin Heidelberg: Springer; 2009.

Gao HX, Sengupta J, Valle M, Korostelev A, Eswar N, Stagg SM, Van Roey P, Agrawal RK, Harvey SC, Sali A, et al. Study of the structural dynamics of the E-coli 70S ribosome using real-space refinement. Cell. 2003;113:789–801. PubMed

Vila-Sanjurjo A, Ridgeway WK, Seymaner V, Zhang W, Santoso S, Yu K, Cate JHD. X-ray crystal structures of the WT and a hyper-accurate ribosome from Escherichia coli. Proc. Natl Acad. Sci. USA. 2003;100:8682–8687. PubMed PMC

Chu VB, Bai Y, Lipfert J, Herschlag D, Doniach S. A repulsive field: advances in the electrostatics of the ion atmosphere. Curr. Opin. Chem. Biol. 2008;12:619–625. PubMed PMC

Chen AA, Draper DE, Pappu RV. Molecular simulation studies of monovalent counterion-mediated interactions in a model RNA kissing loop. J. Mol. Biol. 2009;390:805–819. PubMed PMC

Mitra K, Schaffitzel C, Fabiola F, Chapman MS, Ban N, Frank J. Elongation arrest by SecM via a cascade of ribosomal RNA rearrangements. Mol. Cell. 2006;22:533–543. PubMed

Gutell RR, Gray MW, Schnare MN. A Compilation of Large Subunit (23s-Like and 23s-Like) Ribosomal-Rna Structures - 1993. Nucleic Acids Res. 1993;21:3055–3074. PubMed PMC

Goody TA, Melcher SE, Norman DG, Lilley DMJ. The kink-turn motif in RNA is dimorphic, and metal ion-dependent. RNA. 2004;10:254–264. PubMed PMC

Liu J, Lilley DMJ. The role of specific 2′-hydroxyl groups in the stabilization of the folded conformation of kink-turn RNA. RNA. 2007;13:200–210. PubMed PMC

Matsumura S, Ikawa Y, Inoue T. Biochemical characterization of the kink-turn RNA motif. Nucleic Acids Res. 2003;31:5544–5551. PubMed PMC

Szewczak LBW, Gabrielsen JS, DeGregorio SJ, Strobel SA, Steitz JA. Molecular basis for RNA kink-turn recognition by the h15.5K small RNP protein. RNA. 2005;11:1407–1419. PubMed PMC

Turner B, Lilley DMJ. The importance of G · A hydrogen bonding in the metal ion- and protein-induced folding of a kink turn RNA. J. Mol. Biol. 2008;381:431–442. PubMed

Dixit SB, Beveridge DL, Case DA, Cheatham TE, Giudice E, Lankas F, Lavery R, Maddocks JH, Osman R, Sklenar H, et al. Molecular dynamics simulations of the 136 unique tetranucleotide sequences of DNA oligonucleotides. II: Sequence context effects on the dynamical structures of the 10 unique dinucleotide steps. Biophys. J. 2005;89:3721–3740. PubMed PMC

Cheatham TE, III, Miller JL, Fox T, Darden TA, Kollman PA. Molecular-dynamics simulations on solvated biomolecular systems - the particle mesh ewald method leads to stable trajectories of DNA, RNA, and proteins. J. Am. Chem. Soc. 1995;117:4193–4194.

Perez A, Luque FJ, Orozco M. Dynamics of B-DNA on the microsecond time scale. J. Am. Chem. Soc. 2007;129:14739–14745. PubMed

Ponomarev SY, Thayer KM, Beveridge DL. Ion motions in molecular dynamics simulations on DNA. Proc. Natl Acad. Sci. USA. 2004;101:14771–14775. PubMed PMC

Spackova N, Berger I, Sponer J. Nanosecond molecular dynamics simulations of parallel and antiparallel guanine quadruplex DNA molecules. J. Am. Chem. Soc. 1999;121:5519–5534.

Perez A, Marchan I, Svozil D, Sponer J, Cheatham T.E., 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

Stefl R, Cheatham TE, Spackova N, Fadrna E, Berger I, Koca J, Sponer J. Formation pathways of a guanine-quadruplex DNA revealed by molecular dynamics and thermodynamic analysis of the substates. Biophys. J. 2003;85:1787–1804. PubMed PMC

Krasovska MV, Sefcikova J, Reblova K, Schneider B, Walter NG, Sponer J. Cations and hydration in catalytic RNA: Molecular dynamics of the hepatitis delta virus ribozyme. Biophys. J. 2006;91:626–638. PubMed PMC

Reblova K, Spackova N, Koca J, Leontis NB, Sponer J. Long-residency hydration, cation binding, and dynamics of loop E/helix IV rRNA-L25 protein complex. Biophys. J. 2004;87:3397–3412. PubMed PMC

Reblova K, Spackova N, Sponer JE, Koca J, Sponer J. Molecular dynamics simulations of RNA kissing-loop motifs reveal structural dynamics and formation of cation-binding pockets. Nucleic Acids Res. 2003;31:6942–6952. PubMed PMC

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

Sponer J, Spackova N. Molecular dynamics simulations and their application to four-stranded DNA. Methods. 2007;43:278–290. PubMed PMC

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

Fadrna E, Spackova N, Sarzynska J, Koca J, Orozco M, Cheatham T.E., 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

Kiparisov S, Petrov A, Meskauskas A, Sergiev PV, Dontsova OA, Dinman JD. Structural and functional analysis of 5S rRNA in Saccharomyces cerevisiae. Mol. Genet. Genomics. 2005;274:235–247. PubMed PMC

RNA Structural Dynamics As Captured by Molecular Simulations: A Comprehensive Overview

Structure and mechanical properties of the ribosomal L1 stalk three-way junction

Performance of Molecular Mechanics Force Fields for RNA Simulations: Stability of UUCG and GNRA Hairpins

Quantum chemical studies of nucleic acids: can we construct a bridge to the RNA structural biology and bioinformatics communities?

Molecular dynamics simulations suggest that RNA three-way junctions can act as flexible RNA structural elements in the ribosome

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