Visualization of release factor 3 on the ribosome during termination of protein synthesis

Termination of protein synthesis by the ribosome requires two release factor (RF) classes. The class II RF3 is a GTPase that removes class I RFs (RF1 or RF2) from the ribosome after release of the nascent polypeptide. RF3 in the GDP state binds to the ribosomal class I RF complex, followed by an exchange of GDP for GTP and release of the class I RF. As GTP hydrolysis triggers release of RF3 (ref. 4), we trapped RF3 on Escherichia coli ribosomes using a nonhydrolysable GTP analogue. Here we show by cryo-electron microscopy that the complex can adopt two different conformational states. In ‘state 1’, RF3 is pre-bound to the ribosome, whereas in ‘state 2’ RF3 contacts the ribosome GTPase centre. The transfer RNA molecule translocates from the peptidyl site in state 1 to the exit site in state 2. This translocation is associated with a large conformational rearrangement of the ribosome. Because state 1 seems able to accommodate simultaneously both RF3 and RF2, whose position is known from previous studies, we can infer the release mechanism of class I RFs.

[1]  M. Heel,et al.  Single-particle electron cryo-microscopy: towards atomic resolution , 2000, Quarterly Reviews of Biophysics.

[2]  J. Yuan,et al.  Towards atomic resolution EELS of anisotropic materials , 1999 .

[3]  J. Frank,et al.  A cryo-electron microscopic study of ribosome-bound termination factor RF2 , 2003, Nature.

[4]  M. Ehrenberg,et al.  Stop codon recognition and interactions with peptide release factor RF3 of truncated and chimeric RF1 and RF2 from Escherichia coli , 2003, Molecular microbiology.

[5]  J. Wang,et al.  The crystal structure of elongation factor G complexed with GDP, at 2.7 A resolution. , 1994, The EMBO journal.

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

[7]  A. Avarsson.  Structure-based sequence alignment of elongation factors Tu and G with related GTPases involved in translation. , 1995, Journal of molecular evolution.

[8]  M van Heel,et al.  A new generation of the IMAGIC image processing system. , 1996, Journal of structural biology.

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

[10]  Måns Ehrenberg,et al.  Structure of the Escherichia coli ribosomal termination complex with release factor 2 , 2003, Nature.

[11]  R J Read,et al.  Crystallography & NMR system: A new software suite for macromolecular structure determination. , 1998, Acta crystallographica. Section D, Biological crystallography.

[12]  R. Henderson,et al.  Optimal determination of particle orientation, absolute hand, and contrast loss in single-particle electron cryomicroscopy. , 2003, Journal of molecular biology.

[13]  M van Heel,et al.  The Escherichia coli large ribosomal subunit at 7.5 A resolution. , 1999, Structure.

[14]  A. Liljas,et al.  Three‐dimensional structure of the ribosomal translocase: elongation factor G from Thermus thermophilus. , 1994, The EMBO journal.

[15]  M. Ehrenberg,et al.  Release of peptide promoted by the GGQ motif of class 1 release factors regulates the GTPase activity of RF3. , 2002, Molecular cell.

[16]  P. Kelly,et al.  Release factor RF-3 GTPase activity acts in disassembly of the ribosome termination complex. , 1998, RNA.

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

[18]  H. Noller,et al.  Orientation of Ribosome Recycling Factor in the Ribosome from Directed Hydroxyl Radical Probing , 2002, Cell.

[19]  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.

[20]  Olivier Poch,et al.  PipeAlign: a new toolkit for protein family analysis , 2003, Nucleic Acids Res..

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

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

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

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

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

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

[27]  J. Zou,et al.  Improved methods for building protein models in electron density maps and the location of errors in these models. , 1991, Acta crystallographica. Section A, Foundations of crystallography.

[28]  A. Liljas,et al.  Structure of a mutant EF-G reveals domain III and possibly the fusidic acid binding site. , 2000, Journal of molecular biology.

[29]  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.