Single ribosome dynamics and the mechanism of translation.

Our current understanding of the mechanism of translation is based on nearly fifty years of biochemical and biophysical studies. This mechanism, which requires the ribosome to manipulate tRNA and step repetitively along the mRNA, implies movement. High-resolution structures of the ribosome and its ligands have recently described translation in atomic detail, capturing the endpoints of large-scale rearrangements of the ribosome. Direct observation of the dynamic events that underlie the mechanism of translation is challenged by ensemble averaging in bulk solutions. Single-molecule methods, which eliminate these averaging effects, have emerged as powerful tools to probe the mechanism of translation. Single-molecule fluorescence experiments have described the dynamic motion of the ribosome and tRNA. Single-molecule force measurements have directly probed the forces stabilizing ribosomal complexes. Recent developments have allowed real-time observation of ribosome movement and dynamics during translation. This review covers the contributions of single-molecule studies to our understanding of the dynamic nature of translation.

[1]  H. Craighead,et al.  Zero-mode waveguides: sub-wavelength nanostructures for single molecule studies at high concentrations. , 2008, Methods.

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

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

[4]  Ignacio Tinoco,et al.  Following translation by single ribosomes one codon at a time , 2008, Nature.

[5]  O. Maaløe,et al.  Regulation of the Protein-Synthesizing Machinery—Ribosomes, tRNA, Factors, and So On , 1979 .

[6]  Harry F. Noller,et al.  Transfer RNA shields specific nucleotides in 16S ribosomal RNA from attack by chemical probes , 1986, Cell.

[7]  M. Kozak Initiation of translation in prokaryotes and eukaryotes. , 1999, Gene.

[8]  R. L. Gonzalez,et al.  Translation factors direct intrinsic ribosome dynamics during translation termination and ribosome recycling , 2009, Nature Structural &Molecular Biology.

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

[10]  J. Puglisi,et al.  Solution structure of the A loop of 23S ribosomal RNA , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[11]  J. Nilsson,et al.  Elongation factors on the ribosome. , 2005, Current opinion in structural biology.

[12]  Sotaro Uemura,et al.  Peptide bond formation destabilizes Shine–Dalgarno interaction on the ribosome , 2007, Nature.

[13]  S. Blanchard Single-molecule observations of ribosome function. , 2009, Current opinion in structural biology.

[14]  T. Earnest,et al.  Crystal Structure of the Ribosome at 5.5 Å Resolution , 2001, Science.

[15]  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.

[16]  C. Gualerzi,et al.  Mapping the fMet‐tRNAfMet binding site of initiation factor IF2 , 2000, The EMBO journal.

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

[18]  J. Puglisi,et al.  Thiostrepton inhibition of tRNA delivery to the ribosome. , 2007, RNA.

[19]  D. Steel,et al.  Application of single-molecule spectroscopy in studying enzyme kinetics and mechanism. , 2008, Methods in enzymology.

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

[21]  M. Ehrenberg,et al.  Termination of translation: interplay of mRNA, rRNAs and release factors? , 2003, The EMBO journal.

[22]  M. Rodnina,et al.  Fidelity of aminoacyl-tRNA selection on the ribosome: kinetic and structural mechanisms. , 2001, Annual review of biochemistry.

[23]  Carlos Bustamante,et al.  Recent advances in optical tweezers. , 2008, Annual review of biochemistry.

[24]  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.

[25]  Carlos Bustamante,et al.  Differential detection of dual traps improves the spatial resolution of optical tweezers. , 2006, Proceedings of the National Academy of Sciences of the United States of America.

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

[27]  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.

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

[29]  M. S. Chapman,et al.  Study of the Structural Dynamics of the E. coli 70S Ribosome Using Real-Space Refinement , 2003, Cell.

[30]  Colin Echeverría Aitken,et al.  Translation at the single-molecule level. , 2008, Annual review of biochemistry.

[31]  Graham T Dempsey,et al.  Single-molecule structural dynamics of EF-G--ribosome interaction during translocation. , 2007, Biochemistry.

[32]  Måns Ehrenberg,et al.  Splitting of the posttermination ribosome into subunits by the concerted action of RRF and EF-G. , 2005, Molecular cell.

[33]  G. Wagner,et al.  Translation initiation: structures, mechanisms and evolution , 2004, Quarterly Reviews of Biophysics.

[34]  A. Kapanidis,et al.  Biology, one molecule at a time. , 2009, Trends in biochemical sciences.

[35]  M. Rodnina,et al.  Mechanisms of elongation on the ribosome: dynamics of a macromolecular machine. , 2004, Biochemical Society transactions.

[36]  Måns Ehrenberg,et al.  How initiation factors tune the rate of initiation of protein synthesis in bacteria , 2006, The EMBO journal.

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

[38]  H. Taguchi,et al.  Single-molecule imaging of full protein synthesis by immobilized ribosomes , 2008, Nucleic acids research.

[39]  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.

[40]  M Grunberg-Manago,et al.  Light-scattering studies showing the effect of initiation factors on the reversible dissociation of Escherichia coli ribosomes. , 1975, Journal of molecular biology.

[41]  C. Bustamante,et al.  Overstretching B-DNA: The Elastic Response of Individual Double-Stranded and Single-Stranded DNA Molecules , 1996, Science.

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

[43]  James B. Munro,et al.  A new view of protein synthesis: mapping the free energy landscape of the ribosome using single-molecule FRET. , 2008, Biopolymers.

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

[45]  M. Ehrenberg,et al.  How initiation factors maximize the accuracy of tRNA selection in initiation of bacterial protein synthesis. , 2006, Molecular cell.

[46]  Joachim Frank,et al.  The Cryo-EM Structure of a Translation Initiation Complex from Escherichia coli , 2005, Cell.

[47]  Bruno P. Klaholz,et al.  Structure of the 30S translation initiation complex , 2008, Nature.

[48]  Jon R Lorsch,et al.  The molecular mechanics of eukaryotic translation. , 2003, Annual review of biochemistry.

[49]  M. Ehrenberg,et al.  Release factor RF3 in E.coli accelerates the dissociation of release factors RF1 and RF2 from the ribosome in a GTP‐dependent manner , 1997, The EMBO journal.

[50]  D. P. Fromm,et al.  Methods of single-molecule fluorescence spectroscopy and microscopy , 2003 .

[51]  C. Gualerzi,et al.  Conformational transition of initiation factor 2 from the GTP- to GDP-bound state visualized on the ribosome , 2005, Nature Structural &Molecular Biology.

[52]  W. Greenleaf,et al.  High-resolution, single-molecule measurements of biomolecular motion. , 2007, Annual review of biophysics and biomolecular structure.

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

[54]  M. Ehrenberg,et al.  Ribosome formation from subunits studied by stopped-flow and Rayleigh light scattering , 2004, Biological Procedures Online.

[55]  Colin Echeverría Aitken,et al.  GTP hydrolysis by IF2 guides progression of the ribosome into elongation. , 2009, Molecular cell.

[56]  A. Spirin,et al.  Factor-free ("non-enzymic") and factor-dependent systems of translation of polyuridylic acid by Escherichia coli ribosomes. , 1976, Journal of molecular biology.

[57]  J. Puglisi,et al.  Recognition of the codon-anticodon helix by ribosomal RNA. , 1999, Science.

[58]  B. S. Laursen,et al.  Initiation of Protein Synthesis in Bacteria , 2005, Microbiology and Molecular Biology Reviews.

[59]  J. Puglisi,et al.  tRNA selection and kinetic proofreading in translation , 2004, Nature Structural &Molecular Biology.

[60]  J. Puglisi,et al.  The role of fluctuations in tRNA selection by the ribosome , 2007, Proceedings of the National Academy of Sciences.

[61]  J. Ravel,et al.  The effects of initiation factors IF‐1 and IF‐3 on the dissociation of escherichia coli 70 S ribosomes , 1979, FEBS letters.

[62]  H. Noller,et al.  Catalysis of Ribosomal Translocation by Sparsomycin , 2003, Science.

[63]  J. Frank,et al.  RF3 Induces Ribosomal Conformational Changes Responsible for Dissociation of Class I Release Factors , 2007, Cell.

[64]  A. Spirin,et al.  Translocation makes the ribosome less compact. , 1987, Journal of molecular biology.

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

[66]  M. Ehrenberg,et al.  A Posttermination Ribosomal Complex Is the Guanine Nucleotide Exchange Factor for Peptide Release Factor RF3 , 2001, Cell.

[67]  E. Youngman,et al.  Peptide release on the ribosome: mechanism and implications for translational control. , 2008, Annual review of microbiology.

[68]  Ruben L. Gonzalez,et al.  Site-specific labeling of the ribosome for single-molecule spectroscopy , 2005, Nucleic acids research.

[69]  J Frank,et al.  Domain motions of EF-G bound to the 70S ribosome: insights from a hand-shaking between multi-resolution structures. , 2000, Biophysical journal.

[70]  Hani S. Zaher,et al.  Fidelity at the Molecular Level: Lessons from Protein Synthesis , 2009, Cell.

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

[72]  Scott C. Blanchard,et al.  Approaching translation at atomic resolution , 2000, Nature Structural Biology.

[73]  T. Steitz,et al.  Formation of the First Peptide Bond: The Structure of EF-P Bound to the 70S Ribosome , 2009, Science.

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

[75]  H. Stark,et al.  GTPase Mechanisms and Functions of Translation Factors on the Ribosome , 2000, Biological chemistry.

[76]  A. Spirin,et al.  Non-enzymatic translocation in ribosomes from streptomycin-resistant mutants of Escherichia coli , 1975, Molecular and General Genetics MGG.

[77]  J. Puglisi,et al.  Structure of the A Site of Escherichia coli 16S Ribosomal RNA Complexed with an Aminoglycoside Antibiotic , 1996, Science.

[78]  Joseph D. Puglisi,et al.  Irreversible chemical steps control intersubunit dynamics during translation , 2008, Proceedings of the National Academy of Sciences.

[79]  H. Noller,et al.  Accurate translocation of mRNA by the ribosome requires a peptidyl group or its analog on the tRNA moving into the 30S P site. , 2002, Molecular cell.

[80]  C. Joo,et al.  Advances in single-molecule fluorescence methods for molecular biology. , 2008, Annual review of biochemistry.

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

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

[83]  T. Pape,et al.  Complete kinetic mechanism of elongation factor Tu‐dependent binding of aminoacyl‐tRNA to the A site of the E.coli ribosome , 1998, The EMBO journal.

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

[85]  M. Rodnina,et al.  Mechanism of elongation factor G function in tRNA translocation on the ribosome. , 2001, Cold Spring Harbor symposia on quantitative biology.

[86]  H. Noller,et al.  Interaction of elongation factors EF-G and EF-Tu with a conserved loop in 23S RNA , 1988, Nature.

[87]  Zigurts K. Majumdar,et al.  Measurement of internal movements within the 30 S ribosomal subunit using Förster resonance energy transfer. , 2005, Journal of molecular biology.