Structure of ratcheted ribosomes with tRNAs in hybrid states

During protein synthesis, tRNAs and mRNA move through the ribosome between aminoacyl (A), peptidyl (P), and exit (E) sites of the ribosome in a process called translocation. Translocation is accompanied by the displacement of the tRNAs on the large ribosomal subunit toward the hybrid A/P and P/E states and by a rotational movement (ratchet) of the ribosomal subunits relative to one another. So far, the structure of the ratcheted state has been observed only when translation factors were bound to the ribosome. Using cryo-electron microscopy and classification, we show here that ribosomes can spontaneously adopt a ratcheted conformation with tRNAs in their hybrid states. The peptidyl-tRNA molecule in the A/P state, which is visualized here, is not distorted compared with the A/A state except for slight adjustments of its acceptor end, suggesting that the displacement of the A-site tRNA on the 50S subunit is passive and is induced by the 30S subunit rotation. Simultaneous subunit ratchet and formation of the tRNA hybrid states precede and may promote the subsequent rapid and coordinated tRNA translocation on the 30S subunit catalyzed by elongation factor G.

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

[2]  H. Stark,et al.  Spontaneous reverse movement of mRNA-bound tRNA through the ribosome , 2007, Nature Structural &Molecular Biology.

[3]  J. Frank,et al.  Solution Structure of the E. coli 70S Ribosome at 11.5 Å Resolution , 2000, Cell.

[4]  José María Carazo,et al.  Image processing for electron microscopy single-particle analysis using XMIPP , 2008, Nature Protocols.

[5]  Joachim Frank,et al.  Dynamics of EF-G interaction with the ribosome explored by classification of a heterogeneous cryo-EM dataset. , 2004, Journal of structural biology.

[6]  R. Henderson,et al.  Optimal determination of particle orientation, absolute hand, and contrast loss in single-particle electron cryomicroscopy. , 2003, Journal of molecular biology.

[7]  R. Green,et al.  EF-G-independent reactivity of a pre-translocation-state ribosome complex with the aminoacyl tRNA substrate puromycin supports an intermediate (hybrid) state of tRNA binding. , 2004, RNA.

[8]  M. Rodnina,et al.  Hydrolysis of GTP by elongation factor G drives tRNA movement on the ribosome , 1997, Nature.

[9]  W. Wintermeyer,et al.  Binding of the 3′ terminus of tRNA to 23S rRNA in the ribosomal exit site actively promotes translocation. , 1989, The EMBO journal.

[10]  A Leith,et al.  SPIDER and WEB: processing and visualization of images in 3D electron microscopy and related fields. , 1996, Journal of structural biology.

[11]  M. Rodnina,et al.  Control of phosphate release from elongation factor G by ribosomal protein L7/12 , 2005, The EMBO journal.

[12]  M. Rodnina,et al.  GTP consumption of elongation factor Tu during translation of heteropolymeric mRNAs. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

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

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

[15]  S. Kirillov,et al.  Puromycin reaction for the A site-bound peptidyl-tRNA. , 1992, FEBS letters.

[16]  Joachim Frank,et al.  Locking and Unlocking of Ribosomal Motions , 2003, Cell.

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

[18]  A. Spirin.  A model of the functioning ribosome: locking and unlocking of the ribosome subparticles. , 1969, Cold Spring Harbor symposia on quantitative biology.

[19]  Shigeyuki Yokoyama,et al.  Aminoacylation complex structures of leucyl-tRNA synthetase and tRNALeu reveal two modes of discriminator-base recognition , 2005, Nature Structural &Molecular Biology.

[20]  Conrad C. Huang,et al.  UCSF Chimera—A visualization system for exploratory research and analysis , 2004, J. Comput. Chem..

[21]  M. Rodnina,et al.  Transient kinetics, fluorescence, and FRET in studies of initiation of translation in bacteria. , 2007, Methods in enzymology.

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

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

[24]  Harry F Noller,et al.  Intersubunit movement is required for ribosomal translocation , 2007, Proceedings of the National Academy of Sciences.

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

[26]  Wolfgang Wintermeyer,et al.  An elongation factor G-induced ribosome rearrangement precedes tRNA-mRNA translocation. , 2003, Molecular cell.

[27]  M. Rodnina,et al.  Purine bases at position 37 of tRNA stabilize codon-anticodon interaction in the ribosomal A site by stacking and Mg2+-dependent interactions. , 2004, RNA.

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

[29]  H. Noller,et al.  Translocation of tRNA during protein synthesis , 2002, FEBS letters.

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

[31]  Joachim Frank,et al.  SPIRE: the SPIDER reconstruction engine. , 2007, Journal of structural biology.