Structural characterization of mRNA-tRNA translocation intermediates

Cryo-EM analysis of a wild-type Escherichia coli pretranslocational sample has revealed the presence of previously unseen intermediate substates of the bacterial ribosome during the first phase of translocation, characterized by intermediate intersubunit rotations, L1 stalk positions, and tRNA configurations. Furthermore, we describe the domain rearrangements in quantitative terms, which has allowed us to characterize the processivity and coordination of the conformational reorganization of the ribosome, along with the associated changes in tRNA ribosome-binding configuration. The results are consistent with the view of the ribosome as a molecular machine employing Brownian motion to reach a functionally productive state via a series of substates with incremental changes in conformation.

[1]  Joachim Frank,et al.  The process of mRNA–tRNA translocation , 2007, Proceedings of the National Academy of Sciences.

[2]  J. Frank,et al.  Dynamic reorganization of the functionally active ribosome explored by normal mode analysis and cryo-electron microscopy , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[3]  J. Frank,et al.  Elongation in translation as a dynamic interaction among the ribosome, tRNA, and elongation factors EF-G and EF-Tu , 2009, Quarterly Reviews of Biophysics.

[4]  Joachim Frank,et al.  Cryoelectron microscopy structures of the ribosome complex in intermediate states during tRNA translocation , 2011, Proceedings of the National Academy of Sciences.

[5]  Harry F. Noller,et al.  Intermediate states in the movement of transfer RNA in the ribosome , 1989, Nature.

[6]  Zigurts K. Majumdar,et al.  Observation of intersubunit movement of the ribosome in solution using FRET. , 2007, Journal of molecular biology.

[7]  Jake M. Hofman,et al.  Allosteric collaboration between elongation factor G and the ribosomal L1 stalk directs tRNA movements during translation , 2009, Proceedings of the National Academy of Sciences.

[8]  A. Kelley,et al.  Crystal structure of the hybrid state of ribosome in complex with the guanosine triphosphatase release factor 3 , 2011, Proceedings of the National Academy of Sciences.

[9]  Steven Chu,et al.  tRNA dynamics on the ribosome during translation. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[10]  Jamie H. D. Cate,et al.  Structural basis for mRNA and tRNA positioning on the ribosome , 2006, Proceedings of the National Academy of Sciences.

[11]  J. Holton,et al.  Structures of the Bacterial Ribosome at 3.5 Å Resolution , 2005, Science.

[12]  Klaus Schulten,et al.  Structural insights into cognate versus near‐cognate discrimination during decoding , 2011, The EMBO journal.

[13]  J. Cate,et al.  Structures of the Ribosome in Intermediate States of Ratcheting , 2009, Science.

[14]  Jianlin Lei,et al.  Visualization of the hybrid state of tRNA binding promoted by spontaneous ratcheting of the ribosome. , 2008, Molecular cell.

[15]  Leonardo G. Trabuco,et al.  Flexible fitting of atomic structures into electron microscopy maps using molecular dynamics. , 2008, Structure.

[16]  Taekjip Ha,et al.  Following movement of the L1 stalk between three functional states in single ribosomes , 2009, Proceedings of the National Academy of Sciences.

[17]  Zigurts K. Majumdar,et al.  The antibiotic viomycin traps the ribosome in an intermediate state of translocation , 2007, Nature Structural &Molecular Biology.

[18]  Joachim Frank,et al.  Structure and dynamics of a processive Brownian motor: the translating ribosome. , 2010, Annual review of biochemistry.

[19]  A. Spirin.  The Ribosome as a Conveying Thermal Ratchet Machine , 2009, The Journal of Biological Chemistry.

[20]  Wolfgang Wintermeyer,et al.  Structure of ratcheted ribosomes with tRNAs in hybrid states , 2008, Proceedings of the National Academy of Sciences.

[21]  Marina V. Rodnina,et al.  Ribosome dynamics and tRNA movement by time-resolved electron cryomicroscopy , 2010, Nature.

[22]  James B. Munro,et al.  Correlated conformational events in EF-G and the ribosome regulate translocation , 2010, Nature Structural &Molecular Biology.

[23]  Dmitri N. Ermolenko,et al.  mRNA Translocation Occurs During the Second Step of Ribosomal Intersubunit Rotation , 2010, Nature Structural &Molecular Biology.

[24]  B. Cooperman,et al.  Kinetically competent intermediates in the translocation step of protein synthesis. , 2007, Molecular cell.

[25]  G. Herman,et al.  Disentangling conformational states of macromolecules in 3D-EM through likelihood optimization , 2007, Nature Methods.

[26]  Hanqing Liu,et al.  Single-molecule fluorescence measurements of ribosomal translocation dynamics. , 2011, Molecular cell.

[27]  James B. Munro,et al.  A fast dynamic mode of the EF‐G‐bound ribosome , 2010, The EMBO journal.

[28]  A. Spirin.  How does the ribosome work? A hypothesis based on the two subunit construction of the ribosome. , 1968, Currents in modern biology.

[29]  Steven Chu,et al.  Fluctuations of transfer RNAs between classical and hybrid states. , 2007, Biophysical journal.

[30]  Taekjip Ha,et al.  Spontaneous intersubunit rotation in single ribosomes. , 2008, Molecular cell.

[31]  Joachim Frank,et al.  Structures of modified eEF2·80S ribosome complexes reveal the role of GTP hydrolysis in translocation , 2007, The EMBO journal.

[32]  Christian M T Spahn,et al.  Navigating the ribosome's metastable energy landscape. , 2009, Trends in biochemical sciences.

[33]  Vincent B. Chen,et al.  Structures of the Bacterial Ribosome in Classical and Hybrid States of tRNA Binding , 2011, Science.

[34]  M. Bretscher.  Translocation in Protein Synthesis: A Hybrid Structure Model , 1968, Nature.

[35]  Joachim Frank,et al.  A ratchet-like inter-subunit reorganization of the ribosome during translocation , 2000, Nature.

[36]  Nathan O'Connor,et al.  Identification of two distinct hybrid state intermediates on the ribosome. , 2007, Molecular cell.

[37]  Yong-Gui Gao,et al.  The Structure of the Ribosome with Elongation Factor G Trapped in the Posttranslocational State , 2009, Science.

[38]  R. L. Gonzalez,et al.  Coupling of ribosomal L1 stalk and tRNA dynamics during translation elongation. , 2008, Molecular cell.

[39]  J. Ballesta,et al.  Domain movements of elongation factor eEF2 and the eukaryotic 80S ribosome facilitate tRNA translocation , 2004, The EMBO journal.

[40]  James B. Munro,et al.  Spontaneous formation of the unlocked state of the ribosome is a multistep process , 2009, Proceedings of the National Academy of Sciences.

[41]  James B. Munro,et al.  Structure and dynamics of the mammalian ribosomal pretranslocation complex. , 2011, Molecular cell.