Fluctuations of transfer RNAs between classical and hybrid states.

Adjacent transfer RNAs (tRNAs) in the A- and P-sites of the ribosome are in dynamic equilibrium between two different conformations called classical and hybrid states before translocation. Here, we have used single-molecule fluorescence resonance energy transfer to study the effect of Mg(2+) on tRNA dynamics with and without an acetyl group on the A-site tRNA. When the A-site tRNA is not acetylated, tRNA dynamics do not depend on [Mg(2+)], indicating that the relative positions of the substrates for peptide-bond formation are not affected by Mg(2+). In sharp contrast, when the A-site tRNA is acetylated, Mg(2+) lengthens the lifetime of the classical state but does not change the lifetime of the hybrid state. Based on these findings, the classical state resembles a state with direct stabilization of tertiary structure by Mg(2+) ions whereas the hybrid state resembles a state with little Mg(2+)-assisted stabilization. The antibiotic viomycin, a translocation inhibitor, suppresses tRNA dynamics, suggesting that the enhanced fluctuations of tRNAs after peptide-bond formation drive spontaneous attempts at translocation by the ribosome.

[1]  A. Gordus,et al.  Single-molecule transition-state analysis of RNA folding , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[2]  U. Geigenmüller,et al.  Analysis of the puromycin reaction. The ribosomal exclusion principle for AcPhe-tRNA binding re-examined. , 1986, European journal of biochemistry.

[3]  O. Uhlenbeck,et al.  Contribution of the esterified amino acid to the binding of aminoacylated tRNAs to the ribosomal P- and A-sites. , 2004, Biochemistry.

[4]  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.

[5]  Joachim Frank,et al.  Visualization of Trna Movements on the Escherichia coli 70s Ribosome during the Elongation Cycle , 2000, The Journal of cell biology.

[6]  Eduardo A. Groisman,et al.  An RNA Sensor for Intracellular Mg2+ , 2006, Cell.

[7]  J. Modolell,et al.  The inhibition of ribosomal translocation by viomycin. , 1977, European journal of biochemistry.

[8]  Wolfgang Wintermeyer,et al.  Conformational changes of the small ribosomal subunit during elongation factor G-dependent tRNA-mRNA translocation. , 2004, Journal of molecular biology.

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

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

[11]  J. S. Weinger,et al.  Substrate-assisted catalysis of peptide bond formation by the ribosome , 2004, Nature Structural &Molecular Biology.

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

[13]  S. Joseph,et al.  Conformational changes in the ribosome induced by translational miscoding agents. , 2000, Journal of molecular biology.

[14]  K. Nierhaus,et al.  Viomycin favours the formation of 70S ribosome couples , 1978, Molecular and General Genetics MGG.

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

[16]  R. Micura,et al.  Chemical engineering of the peptidyl transferase center reveals an important role of the 2′-hydroxyl group of A2451 , 2005, Nucleic acids research.

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

[18]  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.

[19]  Annette Sievers,et al.  The ribosome as an entropy trap. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[20]  T. Steitz,et al.  A pre-translocational intermediate in protein synthesis observed in crystals of enzymatically active 50S subunits , 2002, Nature Structural Biology.

[21]  H. Noller,et al.  Chloramphenicol, erythromycin, carbomycin and vernamycin B protect overlapping sites in the peptidyl transferase region of 23S ribosomal RNA. , 1987, Biochimie.

[22]  Thomas A Steitz,et al.  Structural insights into peptide bond formation , 2002, Proceedings of the National Academy of Sciences of the United States of America.

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

[24]  Taekjip Ha,et al.  Mg2+-dependent conformational change of RNA studied by fluorescence correlation and FRET on immobilized single molecules , 2002, Proceedings of the National Academy of Sciences of the United States of America.

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

[26]  S. Joseph,et al.  Rapid kinetic analysis of EF-G-dependent mRNA translocation in the ribosome. , 2003, Journal of molecular biology.

[27]  R. Jernigan,et al.  Global ribosome motions revealed with elastic network model. , 2004, Journal of structural biology.

[28]  Mark Bates,et al.  Short-range spectroscopic ruler based on a single-molecule optical switch. , 2005, Physical review letters.

[29]  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.

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

[31]  M. Heilemann,et al.  Carbocyanine dyes as efficient reversible single-molecule optical switch. , 2005, Journal of the American Chemical Society.