Structures of the Ribosome in Intermediate States of Ratcheting

Translational Rearrangements Conformational changes in the ribosome are required to translocate messenger RNA and transfer RNA (tRNA) during protein biosynthesis. For example, after peptide bond formation, rotation of the large and small subunits results in a hybrid state of tRNA binding—tRNAs are bound respectively in the aminoacyl-tRNA (A) and peptidyl-tRNA (P) sites in the small subunit, but in the P and exit-tRNA (E) sites on the large subunit. Zhang et al. (p. 1014) now describe x-ray structures of the intact Escherichia coli ribosome, either in the apo form or with one or two anticodon stem-loop tRNA mimics bound, which show intermediate states of intersubunit rotation. The structures provide insight into how the interface between the large and small subunits rearranges in discrete steps to reach the hybrid state. Structures of the Escherichia coli 70S ribosome show how the large and small subunits rotate to facilitate protein synthesis. Protein biosynthesis on the ribosome requires repeated cycles of ratcheting, which couples rotation of the two ribosomal subunits with respect to each other, and swiveling of the head domain of the small subunit. However, the molecular basis for how the two ribosomal subunits rearrange contacts with each other during ratcheting while remaining stably associated is not known. Here, we describe x-ray crystal structures of the intact Escherichia coli ribosome, either in the apo-form (3.5 angstrom resolution) or with one (4.0 angstrom resolution) or two (4.0 angstrom resolution) anticodon stem-loop tRNA mimics bound, that reveal intermediate states of intersubunit rotation. In the structures, the interface between the small and large ribosomal subunits rearranges in discrete steps along the ratcheting pathway. Positioning of the head domain of the small subunit is controlled by interactions with the large subunit and with the tRNA bound in the peptidyl-tRNA site. The intermediates observed here provide insight into how tRNAs move into the hybrid state of binding that precedes the final steps of mRNA and tRNA translocation.

[1]  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.

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

[3]  H. Noller,et al.  EF‐G‐catalyzed translocation of anticodon stem–loop analogs of transfer RNA in the ribosome , 1998, The EMBO journal.

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

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

[6]  H. Noller,et al.  Creating ribosomes with an all-RNA 30S subunit P site. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[7]  J. Ballesta,et al.  Domain movements of elongation factor eEF2 and the eukaryotic 80S ribosome facilitate tRNA translocation , 2004, The EMBO journal.

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

[9]  H. Noller,et al.  mRNA Helicase Activity of the Ribosome , 2005, Cell.

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

[11]  R. Green,et al.  Multiple effects of S13 in modulating the strength of intersubunit interactions in the ribosome during translation. , 2005, Journal of molecular biology.

[12]  S. Douthwaite,et al.  Capreomycin binds across the ribosomal subunit interface using tlyA-encoded 2'-O-methylations in 16S and 23S rRNAs. , 2006, Molecular cell.

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

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

[15]  Jamie H. D. Cate,et al.  Structural basis for mRNA and tRNA positioning on the ribosome , 2006, Proceedings of the National Academy of Sciences.

[16]  J. Holton,et al.  A steric block in translation caused by the antibiotic spectinomycin. , 2007, ACS chemical biology.

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

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

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

[20]  B. Cooperman,et al.  Kinetically competent intermediates in the translocation step of protein synthesis. , 2007, Molecular cell.

[21]  Joachim Frank,et al.  The process of mRNA–tRNA translocation , 2007, Proceedings of the National Academy of Sciences.

[22]  Shigeyuki Yokoyama,et al.  Structural basis for interaction of the ribosome with the switch regions of GTP-bound elongation factors. , 2007, Molecular cell.

[23]  H. Noller,et al.  Structural basis for translation termination on the 70S ribosome , 2008, Nature.

[24]  Jianyu Zhu,et al.  Crystal structure of a translation termination complex formed with release factor RF2 , 2008, Proceedings of the National Academy of Sciences.

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

[26]  Sabine Petry,et al.  Insights into Translational Termination from the Structure of RF2 Bound to the Ribosome , 2008, Science.

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

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

[29]  I. Izquierdo,et al.  Dopamine Controls Persistence of Long-Term Memory Storage , 2009, Science.