Visualization of Trna Movements on the Escherichia coli 70s Ribosome during the Elongation Cycle

Three-dimensional cryomaps have been reconstructed for tRNA–ribosome complexes in pre- and posttranslocational states at 17-Å resolution. The positions of tRNAs in the A and P sites in the pretranslocational complexes and in the P and E sites in the posttranslocational complexes have been determined. Of these, the P-site tRNA position is the same as seen earlier in the initiation-like fMet-tRNAf Met-ribosome complex, where it was visualized with high accuracy. Now, the positions of the A- and E-site tRNAs are determined with similar accuracy. The positions of the CCA end of the tRNAs at the A site are different before and after peptide bond formation. The relative positions of anticodons of P- and E-site tRNAs in the posttranslocational state are such that a codon–anticodon interaction at the E site appears feasible.

[1]  W. Wintermeyer,et al.  Affinities of tRNA binding sites of ribosomes from Escherichia coli. , 1986, Biochemistry.

[2]  K. Nierhaus,et al.  Kinetic and thermodynamic parameters for tRNA binding to the ribosome and for the translocation reaction. , 1992, The Journal of biological chemistry.

[3]  K. Nierhaus,et al.  Spontaneous, elongation factor G independent translocation of Escherichia coli ribosomes. , 1983, The Journal of biological chemistry.

[4]  H. Rheinberger,et al.  Three tRNA binding sites on Escherichia coli ribosomes. , 1981, Proceedings of the National Academy of Sciences of the United States of America.

[5]  Y. Ivanov,et al.  70-S ribosomes of Escherichia coli have an additional site for deacylated tRNA binding. , 2005, European journal of biochemistry.

[6]  K. Nierhaus,et al.  Solution of the ribosome riddle: how the ribosome selects the correct aminoacyl‐tRNA out of 41 similar contestants , 1993, Molecular microbiology.

[7]  J. Frank,et al.  Conformational variability in Escherichia coli 70S ribosome as revealed by 3D cryo-electron microscopy. , 1999, The international journal of biochemistry & cell biology.

[8]  E. Dabbs,et al.  Functional studies on ribosomes lacking protein L1 from mutant Escherichia coli. , 1980, European journal of biochemistry.

[9]  J Frank,et al.  Visualization of elongation factor G on the Escherichia coli 70S ribosome: the mechanism of translocation. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[10]  A. Gnirke,et al.  tRNA binding sites on the subunits of Escherichia coli ribosomes. , 1986, The Journal of biological chemistry.

[11]  V. Ramakrishnan,et al.  Structure of a bacterial 30S ribosomal subunit at 5.5 Å resolution , 1999, Nature.

[12]  Harry F Noller,et al.  Molecular Movement inside the Translational Engine , 1998, Cell.

[13]  R. Brimacombe,et al.  Arrangement of tRNAs in Pre- and Posttranslocational Ribosomes Revealed by Electron Cryomicroscopy , 1997, Cell.

[14]  J. Wower,et al.  A Model of the tRNA Binding Sites on the Escherichia Coli Ribosome , 1993 .

[15]  U. Geigenmüller,et al.  Significance of the third tRNA binding site, the E site, on E. coli ribosomes for the accuracy of translation: an occupied E site prevents the binding of non‐cognate aminoacyl‐tRNA to the A site. , 1990, The EMBO journal.

[16]  C. Cantor,et al.  Distance moved by transfer RNA during translocation from the A site to the P site on the ribosome. , 1982, Journal of molecular biology.

[17]  T. Earnest,et al.  X-ray crystal structures of 70S ribosome functional complexes. , 1999, Science.

[18]  J. Frank The ribosome-structure and functional ligand-binding experiments using cryo-electron microscopy. , 1998, Journal of structural biology.

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

[20]  U. Bommer Ribosomes and polysomes , 1997 .

[21]  Roger A. Garrett,et al.  The Ribosome, Structure, Function, Antibiotics, and Cellular Interactions , 2000 .

[22]  J. Frank,et al.  The ribosome at improved resolution: new techniques for merging and orientation refinement in 3D cryo-electron microscopy of biological particles. , 1994, Ultramicroscopy.

[23]  J. Dubochet,et al.  Cryo-electron microscopy of vitrified specimens , 1988, Quarterly Reviews of Biophysics.

[24]  J Frank,et al.  Three-dimensional reconstruction of the Escherichia coli 30 S ribosomal subunit in ice. , 1996, Journal of molecular biology.

[25]  J. Frank,et al.  Structural studies of the translational apparatus. , 1999, Current opinion in structural biology.

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

[27]  J Frank,et al.  Electron microscopy and computer image averaging of ice-embedded large ribosomal subunits from Escherichia coli. , 1988, Journal of molecular biology.

[28]  R. Agrawal,et al.  Sites of Ribosomal RNAs Involved in the Subunit Association of Tight and Loose Couple Ribosomes* , 1996, The Journal of Biological Chemistry.

[29]  J. Frank,et al.  A model of protein synthesis based on cryo-electron microscopy of the E. coli ribosome , 1995, Nature.

[30]  J. Frank,et al.  Direct Visualization of A-, P-, and E-Site Transfer RNAs in the Escherichia coli Ribosome , 1996, Science.

[31]  E. Makarov,et al.  Quantitative study of interaction of deacylated tRNA with Escherichia coli ribosomes , 1983, FEBS letters.

[32]  H. Wittmann RIBOSOMES: STRUCTURE, FUNCTION AND EVOLUTION , 1976 .

[33]  V. Lim,et al.  Analysis of interactions between the codon-anticodon duplexes within the ribosome: their role in translation. , 1997, Journal of molecular biology.

[34]  K. Nierhaus The allosteric three-site model for the ribosomal elongation cycle: features and future. , 1990, Biochemistry.

[35]  R. Brimacombe,et al.  The structure of ribosomal RNA: a three-dimensional jigsaw puzzle. , 1995, European journal of biochemistry.

[36]  P. Moore,et al.  An investigation of the conformational properties of ribosomes using N-ethylmaleimide as a probe. , 1979, European journal of biochemistry.

[37]  J. Frank,et al.  The movement of tRNA through the ribosome. , 1998, Biophysical journal.

[38]  Joachim Frank,et al.  EF-G-dependent GTP hydrolysis induces translocation accompanied by large conformational changes in the 70S ribosome , 1999, Nature Structural Biology.

[39]  A. P. Potapov,et al.  Synergism between the GTPase activities of EF-Tu.GTP and EF-G.GTP on empty ribosomes. Elongation factors as stimulators of the ribosomal oscillation between two conformations. , 1994, Journal of molecular biology.

[40]  J. Frank,et al.  Studies of Elongation Factor G-Dependent tRNA Translocation by Three-Dimensional Cryo-Electron Microscopy , 2000 .

[41]  J. Frank,et al.  Effect of Buffer Conditions on the Position of tRNA on the 70 S Ribosome As Visualized by Cryoelectron Microscopy* , 1999, The Journal of Biological Chemistry.

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

[43]  E Westhof,et al.  Restrained refinement of two crystalline forms of yeast aspartic acid and phenylalanine transfer RNA crystals. , 1987, Acta crystallographica. Section A, Foundations of crystallography.

[44]  J. Frank,et al.  Three-dimensional reconstruction with contrast transfer function correction from energy-filtered cryoelectron micrographs: procedure and application to the 70S Escherichia coli ribosome. , 1997, Journal of structural biology.

[45]  K. Nierhaus,et al.  Self-coded 3′-Extension of Run-off Transcripts Produces Aberrant Products during in Vitro Transcription with T7 RNA Polymerase (*) , 1995, The Journal of Biological Chemistry.

[46]  R. Green,et al.  The ribosome revealed , 1999, Nature Structural Biology.

[47]  H. Wittmann THE ESCHERICHIA COLI RIBOSOME , 1979 .

[48]  Transfer RNA docking pair model in the ribosomal pre- and post-translocational states. , 1997, Nucleic acids research.

[49]  F. Zemlin,et al.  Structure of keyhole limpet hemocyanin type 1 (KLH1) at 15 A resolution by electron cryomicroscopy and angular reconstitution. , 1997, Journal of molecular biology.

[50]  H. Noller,et al.  Ribosomes and translation. , 1997, Annual review of biochemistry.

[51]  K. Nierhaus,et al.  Structure of the elongating ribosome: arrangement of the two tRNAs before and after translocation. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[52]  C. Spahn,et al.  Protection Patterns of tRNAs Do Not Change during Ribosomal Translocation* , 1998, The Journal of Biological Chemistry.

[53]  W. Wintermeyer,et al.  Topological arrangement of two transfer RNAs on the ribosome. Fluorescence energy transfer measurements between A and P site-bound tRNAphe. , 1983, Journal of molecular biology.

[54]  W. Wintermeyer,et al.  tRNA binding sites of ribosomes from Escherichia coli. , 1984, Biochemistry.

[55]  J Frank,et al.  Escherichia coli 70 S ribosome at 15 A resolution by cryo-electron microscopy: localization of fMet-tRNAfMet and fitting of L1 protein. , 1998, Journal of molecular biology.

[56]  R. Brimacombe,et al.  Visualization of elongation factor Tu on the Escherichia coli ribosome , 1997, Nature.

[57]  R. Brimacombe,et al.  A new model for the three-dimensional folding of Escherichia coli 16 S ribosomal RNA. II. The RNA-protein interaction data. , 1997, Journal of molecular biology.

[58]  Walter E. Hill,et al.  The Ribosome : structure, function, and evolution , 1990 .

[59]  C. Spahn,et al.  Models of the elongation cycle: an evaluation. , 1998, Biological chemistry.

[60]  H. Noller,et al.  Interaction of tRNA with 23S rRNA in the ribosomal A, P, and E sites , 1989, Cell.

[61]  W. Wintermeyer,et al.  Pre-steady-state kinetics of ribosomal translocation. , 1986, Journal of molecular biology.

[62]  C. Spahn,et al.  Are the current three-site models valid descriptions of the ribosomal elongation cycle? , 1997, Proceedings of the National Academy of Sciences of the United States of America.