Ribosomal Dynamics: Intrinsic Instability of a Molecular Machine

Part of the Springer Series in Biophysics book series (BIOPHYSICS, volume 13)

Ribosomes are molecular machines that translate genetic message into nascent peptides, through a complex dynamics interplay with mRNAs, tRNAs, and various protein factors. A prominent example of ribosomal dynamics is the rotation of small ribosomal subunit with respect to a large subunit, characterized as the “ratchet motion,” which is triggered by the binding of several translation factors. Here, we analyze two kinds of ribosomal ratchet motions, induced by the binding of EF-G and RF3, respectively, as previously observed by cryo-electron microscopy. Using the flexible fitting technique (real-space refinement) and an RNA secondary structure display tool (coloRNA), we obtained quasi-atomic models of the ribosome in these ratchet-motion-related functional states and mapped the observed differences onto the highly conserved RNA secondary structure. Comparisons between two sets of ratchet motions revealed that, while the overall patterns of the RNA displacement are very similar, several local regions stand out in their differential behavior, including the highly conserved GAC (GTPase-associated-center) region. We postulate that these regions are important in modulating general ratchet motion and bestowing it with the dynamic characteristics required for the specific function.

Keywords

Hydrolysis Codon Polypeptide Macromolecule Saccharomyces 

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References

  1. Agrawal RK, Heagle AB, Penczek P, Grassucci RA, Frank J (1999) EF-G-dependent GTP hydrolysis induces translocation accompanied by large conformational changes in the 70S ribosome. Nat Struct Biol 6:643–647CrossRefGoogle Scholar
  2. Allen GS, Zavialov A, Gursky R, Ehrenberg M, Frank J (2005) The cryo-EM structure of a translation initiation complex from Escherichia coli. Cell 121:703–712CrossRefGoogle Scholar
  3. Baker TS, Johnson JE (1996) Low resolution meets high: towards a resolution continuum from cells to atoms. Curr Opin Struct Biol 6:585–594CrossRefGoogle Scholar
  4. Ban N, Nissen P, Hansen J, Moore PB, Steitz TA (2000) The complete atomic structure of the large ribosomal subunit at 2.4 A resolution. Science 289:905–920CrossRefADSGoogle Scholar
  5. Barat C, Datta PP, Raj VS, Sharma MR, Kaji H, Kaji A, Agrawal RK (2007) Progression of the ribosome recycling factor through the ribosome dissociates the two ribosomal subunits. Mol Cell 27:250–261CrossRefGoogle Scholar
  6. Blanchard SC, Gonzalez RL, Kim HD, Chu S, Puglisi JD (2004) tRNA selection and kinetic proofreading in translation. Nat Struct Mol Biol 11:1008–1014CrossRefGoogle Scholar
  7. Borovinskaya MA, Pai RD, Zhang W, Schuwirth BS, Holton JM, Hirokawa G, Kaji H, Kaji A, Cate JH (2007) Structural basis for aminoglycoside inhibition of bacterial ribosome recycling. Nat Struct Mol Biol 14:727–732CrossRefGoogle Scholar
  8. Brunger AT, Adams PD, Clore GM, DeLano WL, Gros P, Grosse-Kunstleve RW, Jiang JS, Nilges N, Pannu NS, Read RJ, Rice LM, Simonson T, Warren GL (1998) Crystallography and NMR system (CNS): a new software system for macromolecular structure determination. Acta Cryst D 54:905–921CrossRefGoogle Scholar
  9. Chapman MS (1995) Restrained real-space macromolecular atomic refinement using a new resolution-dependent electron-density function. Acta Cryst A 51:69–80CrossRefGoogle Scholar
  10. Ermolenko DN, Majumdar ZK, Hickerson RP, Spiegel PC, Clegg RM, Noller HF (2007) Observation of intersubunit movement of the ribosome in solution using FRET. J Mol Biol 370:530–540CrossRefGoogle Scholar
  11. Fabiola F, Chapman MS (2005) Fitting of high-resolution structures into electron microscopy reconstruction images. Structure 13:389–400CrossRefGoogle Scholar
  12. Frank J (2006) Three-dimensional electron microscopy of macromolecular assemblies. Oxford University Press, New YorkCrossRefGoogle Scholar
  13. Frank J, Agrawal RK (2000) A ratchet-like inter-subunit reorganization of the ribosome during translocation. Nature 406:318–322CrossRefADSGoogle Scholar
  14. Frank J, Gao H, Sengupta J, Gao N, Taylor DJ (2007) The process of mRNA-tRNA translocation. Proc Natl Acad Sci USA 104:19671–19678CrossRefADSGoogle Scholar
  15. Fu J, Gao H, Frank J (2007) Unsupervised classification of single particles by cluster tracking in multi-dimensional space. J Struct Biol 157:226–239CrossRefGoogle Scholar
  16. Gao H, Frank J (2005) Molding atomic structures into intermediate-resolution cryo-EM density maps of ribosomal complexes using real-space refinement. Structure 13:401–406CrossRefGoogle Scholar
  17. Gao H, Sengupta J, Valle M, Korostelev A, Eswar N, Stagg SM, Van Roey P, Agrawal RK, Harvey SC, Sali A, Chapman MS, Frank J (2003) Study of the structural dynamics of the E. coli 70S ribosome using real space refinement. Cell 113:789–801CrossRefGoogle Scholar
  18. Gao H, Valle M, Ehrenberg M, Frank J (2004) Dynamics of EF-G interaction with the ribosome explored by classification of a heterogeneous cryo-EM dataset. J Struct Biol 147:283–290CrossRefGoogle Scholar
  19. Gao N, Zavialov AV, Li W, Sengupta J, Valle M, Gursky RP, Ehrenberg M, Frank J (2005) Mechanism for the disassembly of the posttermination complex inferred from cryo-EM studies. Mol Cell 18:663–674CrossRefGoogle Scholar
  20. Gao H, Zhou Z, Rawat U, Huang C, Bouakaz L, Wang C, Cheng Z, Liu Y, Zavialov A, Gursky R, Sanyal S, Ehrenberg M, Frank J, Song H (2007) RF3 induces ribosomal conformational changes responsible for dissociation of class I release factors. Cell 129:929–941CrossRefGoogle Scholar
  21. Grassucci RA, Taylor DJ, Frank J (2007) Preparation of macromolecular complexes for cryo-electron microscopy. Nat Protocols 2:3239–3246CrossRefGoogle Scholar
  22. Grassucci RA, Taylor D, Frank J (2008) Visualization of macromolecular complexes using cryo-electron microscopy with FEI Tecnai transmission electron microscopes. Nat Protocols 3:330–339CrossRefGoogle Scholar
  23. Halic M, Blau M, Becker T, Mielke T, Pool MR, Wild K, Sinning I, Beckmann R (2006) Following the signal sequence from ribosomal tunnel exit to signal recognition particle. Nature 444:507–511CrossRefADSGoogle Scholar
  24. Harms J, Schlunzen F, Zarivach R, Bashan A, Gat S, Agmon I, Bartels H, Franceschi F, Yonath A (2001) High resolution structure of the large ribosomal subunit from a mesophilic eubacterium. Cell 107:679–688CrossRefGoogle Scholar
  25. Klaholz BP, Myasnikov AG, van Heel M (2004) Visualization of release factor 3 on the ribosome during termination of protein synthesis. Nature 427:862–865CrossRefADSGoogle Scholar
  26. Korostelev A, Trakhanov S, Laurberg M, Noller HF (2006) Crystal structure of a 70S ribosome-tRNA complex reveals functional interactions and rearrangements. Cell 126:1065–1077CrossRefGoogle Scholar
  27. LeBarron J, Mitra K, Frank J (2007) Displaying 3D data on RNA secondary structures: coloRNA. J Struct Biol 157:262–270CrossRefGoogle Scholar
  28. Munro JB, Altman RB, O'Connor N, Blanchard SC (2007) Identification of two distinct hybrid state intermediates on the ribosome. Mol Cell 25:505–517CrossRefGoogle Scholar
  29. Petry S, Brodersen DE, Murphy FV, Dunham CM, Selmer M, Tarry MJ, Kelley AC, Ramakrishnan V (2005) Crystal structures of the ribosome in complex with release factors RF1 and RF2 bound to a cognate stop codon. Cell 123:1255–1266CrossRefGoogle Scholar
  30. Rawat UBS, Zavialov AV, Sengupta J, Valle M, Grassucci RA, Linde J, Vestergaard B, Ehrenberg M, Frank J (2003) A cryo-electron microscopic study of ribosome-bound termination factor RF2. Nature 421:87–90CrossRefADSGoogle Scholar
  31. Rawat U, Gao H, Zavialov AV, Gursky R, Ehrenberg M, Frank J (2006) Interactions of the release factor RF1 with the ribosome as revealed by cryo-EM. J Mol Biol 357:1144–1153CrossRefGoogle Scholar
  32. Rossmann MG (2000) Fitting atomic models into electron-microscopy maps. Acta Cryst D56:1341–1349Google Scholar
  33. Scheres SH, Gao H, Valle M, Herman GT, Eggermont PP, Frank J, Carazo JM (2007) Disentangling conformational states of macromolecules in 3D-EM through likelihood optimization. Nat Methods 4:27–29CrossRefGoogle Scholar
  34. Schluenzen F, Tocilj A, Zarivach R, Harms J, Gluehmann M, Janell D, Bashan A, Bartels H, Agmon I, Franceschi F, Yonath A (2000) Structure of functionally activated small ribosomal subunit at 3.3 angstroms resolution. Cell 102:615–623CrossRefGoogle Scholar
  35. Schuwirth BS, Borovinskaya MA, Hau CW, Zhang W, Vila-Sanjurjo A, Holton JM, Cate JH (2005) Structures of the bacterial ribosome at 3.5 A resolution. Science 310:827–834CrossRefADSGoogle Scholar
  36. Selmer M, Dunham CM, Murphy FV, Weixlbaumer A, Petry S, Kelley AC, Weir JR, Ramakrishnan V (2006) Structure of the 70S ribosome complexed with mRNA and tRNA. Science 313:1935–1942CrossRefADSGoogle Scholar
  37. Spahn CM, Beckmann E, Eswar N, Penczek PA, Sali A, Blobel G, Frank J (2001) Structure of the 80S ribosome from Saccharomyces cerevisiae — tRNA-ribosome and subunit-subunit interactions. Cell 107:373–386CrossRefGoogle Scholar
  38. Spahn CMT, Gomez-Lorenzo MG, Grassucci GA, Jorgensen R, Andersen GR, Beckmann R, Penczek PA, Ballesta JPG, Frank J (2004) Domain movements of elongation factor eEF2 and the eukaryotic 80S ribosome facilitate tRNA translocation. EMBO J 23:1008–1019CrossRefGoogle Scholar
  39. Spirin AS (1968) How does the ribosome work? A hypothesis based on the two subunit construction of the ribosome. Curr Mod Biol 2:115–127Google Scholar
  40. Spirin AS (2002) Ribosome as a molecular machine. FEBS Lett 514:2–10CrossRefGoogle Scholar
  41. Tama F, Valle M, Frank J, Brooks CL III (2003) Dynamic reorganization of the functionally active ribosome explored by normal mode analysis and cryo-electron microscopy. Proc Natl Acad Sci USA 100:9319–9323CrossRefADSGoogle Scholar
  42. Taylor DJ, Nilsson J, Merrill AR, Andersen GR, Nissen P, Frank J (2007) Structures of modified eEF2 80S ribosome complexes reveal the role of GTP hydrolysis in translocation. EMBO J 26:2421–2431CrossRefGoogle Scholar
  43. Trabnco LG, Villa E, Mitra K, Frank J, Schulten K (2008) Flexible fitting of atomic structures into electron microscopy maps using molecular dynamics. Structure 16:673–683CrossRefGoogle Scholar
  44. Valle M, Sengupta J, Swami K, Grassucci RA, Burkhardt N, Nierhaus KH, Agrawal RK, Frank J (2002) Cryo-EM reveals an active role for the aminoacyl-tRNA in the accommodation process. EMBO J 21:3557–3567CrossRefGoogle Scholar
  45. Valle M, Zavialov AV, Sengupta J, Rawat U, Ehrenberg M, Frank J (2003) Locking and unlocking of ribosomal motions. Cell 114:123–134CrossRefGoogle Scholar
  46. Wang Y, Rader AJ, Bahar I, Jernigan RL (2004) Global ribosome motions revealed with elastic network model. J Struct Biol 147:302–314CrossRefGoogle Scholar
  47. Weixlbaumer A, Petry S, Dunham CM, Selmer M, Kelley AC, Ramakrishnan V (2007) Crystal structure of the ribosome recycling factor bound to the ribosome. Nat Struct Mol Biol 14:733–737CrossRefGoogle Scholar
  48. Wimberly BT, Brodersen DE, Clemons WM Jr., Morgan-Warren RJ, Carter AP, von Rhein C, Hartsch T, Ramakrishnan V (2000) Structure of the 30S ribosomal subunit. Nature 407:327–339CrossRefADSGoogle Scholar
  49. Zavialov AV, Ehrenberg M (2003) Peptidyl-tRNA regulates the GTPase activity of translation factors. Cell 114:113–122CrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2009

Authors and Affiliations

  1. 1.Wadsworth CenterEmpire State PlazaAlbanyUSA
  2. 2.Howard Hughes Medical Institute, Department of Biochemistry and Molecular Biophysics and Department of Biological SciencesColumbia UniversityUSA

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