Irreversible chemical steps control intersubunit dynamics during translation

The ribosome, a two-subunit macromolecular machine, deciphers the genetic code and catalyzes peptide bond formation. Dynamic rotational movement between ribosomal subunits is likely required for efficient and accurate protein synthesis, but direct observation of intersubunit dynamics has been obscured by the repetitive, multistep nature of translation. Here, we report a collection of single-molecule fluorescence resonance energy transfer assays that reveal a ribosomal intersubunit conformational cycle in real time during initiation and the first round of elongation. After subunit joining and delivery of correct aminoacyl-tRNA to the ribosome, peptide bond formation results in a rapid conformational change, consistent with the counterclockwise rotation of the 30S subunit with respect to the 50S subunit implied by prior structural and biochemical studies. Subsequent binding of elongation factor G and GTP hydrolysis results in a clockwise rotation of the 30S subunit relative to the 50S subunit, preparing the ribosome for the next round of tRNA selection and peptide bond formation. The ribosome thus harnesses the free energy of irreversible peptidyl transfer and GTP hydrolysis to surmount activation barriers to large-scale conformational changes during translation. Intersubunit rotation is likely a requirement for the concerted movement of tRNA and mRNA substrates during translocation.

[1]  J. Carbon,et al.  Studies on the thionucleotides in transfer ribonucleic acid. Addition of N-ethylmaleimide and formation of mixed disulfides with thiol compounds. , 1968, Biochemistry.

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

[3]  Y. Mechulam,et al.  Crystallization and preliminary X‐ray analysis of Escherichia coli methionyl‐tRNAMetf formyltransferase complexed with formyl‐methionyl‐tRNAMetf , 1999 .

[4]  W. Wintermeyer,et al.  Effect of translocation on topology and conformation of anticodon and D loops of tRNAPhe. , 1981, Journal of molecular biology.

[5]  A. Yonath,et al.  Antibiotics targeting ribosomes: resistance, selectivity, synergism and cellular regulation. , 2005, Annual review of biochemistry.

[6]  J. Åqvist,et al.  Mechanism of peptide bond synthesis on the ribosome. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

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

[8]  M. Rodnina,et al.  Energetic contribution of tRNA hybrid state formation to translocation catalysis on the ribosome , 2000, Nature Structural Biology.

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

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

[11]  R. Gutell,et al.  Construction and fine mapping of recombinant plasmids containing the rrnB ribosomal RNA operon of E. coli. , 1981, Plasmid.

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

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

[14]  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.

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

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

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

[18]  Frank Schluenzen,et al.  High Resolution Structure of the Large Ribosomal Subunit from a Mesophilic Eubacterium , 2001, Cell.

[19]  M. Heel,et al.  Large-Scale Movement of Elongation Factor G and Extensive Conformational Change of the Ribosome during Translocation , 2000, Cell.

[20]  Joachim Frank,et al.  Cryo‐EM reveals an active role for aminoacyl‐tRNA in the accommodation process , 2002, The EMBO journal.

[21]  V. Ramakrishnan,et al.  Recognition of Cognate Transfer RNA by the 30S Ribosomal Subunit , 2001, Science.

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

[23]  Harry F. Noller,et al.  Crystal Structure of a 70S Ribosome-tRNA Complex Reveals Functional Interactions and Rearrangements , 2014, Cell.

[24]  A. Banerjee,et al.  Mg2+ Is Not Catalytically Required in the Intrinsic and Kirromycin-stimulated GTPase Action of Thermus thermophilus EF-Tu* , 2001, The Journal of Biological Chemistry.

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

[26]  M. Heel,et al.  Ribosome interactions of aminoacyl-tRNA and elongation factor Tu in the codon-recognition complex , 2002, Nature Structural Biology.

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

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

[29]  T. Steitz.  A structural understanding of the dynamic ribosome machine , 2008, Nature Reviews Molecular Cell Biology.

[30]  H. Noller,et al.  A functional pseudoknot in 16S ribosomal RNA. , 1991, The EMBO journal.

[31]  Gilad Haran,et al.  Noise reduction in single-molecule fluorescence trajectories of folding proteins , 2004 .

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

[33]  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.

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

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

[36]  M. Selmer,et al.  Structure of the 70S Ribosome Complexed with mRNA and tRNA , 2006, Science.

[37]  Colin Echeverría Aitken,et al.  An oxygen scavenging system for improvement of dye stability in single-molecule fluorescence experiments. , 2008, Biophysical journal.

[38]  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.

[39]  Måns Ehrenberg,et al.  Peptidyl-tRNA Regulates the GTPase Activity of Translation Factors , 2003, Cell.

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

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

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

[43]  R. A. Kennedy,et al.  Forward-backward non-linear filtering technique for extracting small biological signals from noise , 1991, Journal of Neuroscience Methods.

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