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  • 1
    Publication Date: 2010-07-16
    Description: The translocation step of protein synthesis entails large-scale rearrangements of the ribosome-transfer RNA (tRNA) complex. Here we have followed tRNA movement through the ribosome during translocation by time-resolved single-particle electron cryomicroscopy (cryo-EM). Unbiased computational sorting of cryo-EM images yielded 50 distinct three-dimensional reconstructions, showing the tRNAs in classical, hybrid and various novel intermediate states that provide trajectories and kinetic information about tRNA movement through the ribosome. The structures indicate how tRNA movement is coupled with global and local conformational changes of the ribosome, in particular of the head and body of the small ribosomal subunit, and show that dynamic interactions between tRNAs and ribosomal residues confine the path of the tRNAs through the ribosome. The temperature dependence of ribosome dynamics reveals a surprisingly flat energy landscape of conformational variations at physiological temperature. The ribosome functions as a Brownian machine that couples spontaneous conformational changes driven by thermal energy to directed movement.〈br /〉〈span class="detail_caption"〉Notes: 〈/span〉Fischer, Niels -- Konevega, Andrey L -- Wintermeyer, Wolfgang -- Rodnina, Marina V -- Stark, Holger -- England -- Nature. 2010 Jul 15;466(7304):329-33. doi: 10.1038/nature09206.〈br /〉〈span class="detail_caption"〉Author address: 〈/span〉3D Electron Cryomicroscopy Group, Max Planck Institute for Biophysical Chemistry, Am Fassberg 11, 37077 Gottingen, Germany.〈br /〉〈span class="detail_caption"〉Record origin:〈/span〉 〈a href="http://www.ncbi.nlm.nih.gov/pubmed/20631791" target="_blank"〉PubMed〈/a〉
    Keywords: Cryoelectron Microscopy ; Escherichia coli ; Kinetics ; Models, Molecular ; Molecular Conformation ; *Movement ; *Protein Biosynthesis ; RNA, Transfer/genetics/*metabolism ; Ribosome Subunits, Large, Bacterial/chemistry/metabolism ; Ribosome Subunits, Small, Bacterial/chemistry/metabolism ; Ribosomes/chemistry/*metabolism ; Temperature ; Thermodynamics ; Time Factors
    Print ISSN: 0028-0836
    Electronic ISSN: 1476-4687
    Topics: Biology , Chemistry and Pharmacology , Medicine , Natural Sciences in General , Physics
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  • 2
    Publication Date: 2012-12-15
    Description: Elongation factor P (EF-P) is a translation factor of unknown function that has been implicated in a great variety of cellular processes. Here, we show that EF-P prevents ribosome from stalling during synthesis of proteins containing consecutive prolines, such as PPG, PPP, or longer proline strings, in natural and engineered model proteins. EF-P promotes peptide-bond formation and stabilizes the peptidyl-transfer RNA in the catalytic center of the ribosome. EF-P is posttranslationally modified by a hydroxylated beta-lysine attached to a lysine residue. The modification enhances the catalytic proficiency of the factor mainly by increasing its affinity to the ribosome. We propose that EF-P and its eukaryotic homolog, eIF5A, are essential for the synthesis of a subset of proteins containing proline stretches in all cells.〈br /〉〈span class="detail_caption"〉Notes: 〈/span〉Doerfel, Lili K -- Wohlgemuth, Ingo -- Kothe, Christina -- Peske, Frank -- Urlaub, Henning -- Rodnina, Marina V -- New York, N.Y. -- Science. 2013 Jan 4;339(6115):85-8. doi: 10.1126/science.1229017. Epub 2012 Dec 13.〈br /〉〈span class="detail_caption"〉Author address: 〈/span〉Department of Physical Biochemistry, Max Planck Institute for Biophysical Chemistry, Goettingen, Germany.〈br /〉〈span class="detail_caption"〉Record origin:〈/span〉 〈a href="http://www.ncbi.nlm.nih.gov/pubmed/23239624" target="_blank"〉PubMed〈/a〉
    Keywords: Amino Acid Sequence ; Escherichia coli/genetics/*metabolism ; Lysine/metabolism ; Molecular Sequence Data ; Peptide Elongation Factors/*metabolism ; Proline/genetics/*metabolism ; Protein Biosynthesis ; Protein Processing, Post-Translational ; Ribosomes/*metabolism
    Print ISSN: 0036-8075
    Electronic ISSN: 1095-9203
    Topics: Biology , Chemistry and Pharmacology , Computer Science , Medicine , Natural Sciences in General , Physics
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  • 3
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    American Association for the Advancement of Science (AAAS)
    Publication Date: 2013-07-03
    Description: 〈br /〉〈span class="detail_caption"〉Notes: 〈/span〉Rodnina, Marina V -- New York, N.Y. -- Science. 2013 Jun 28;340(6140):1534-5. doi: 10.1126/science.1240090.〈br /〉〈span class="detail_caption"〉Author address: 〈/span〉Max Planck Institute for Biophysical Chemistry, 37077 Goettingen, Germany. rodnina@mpibpc.mpg.de〈br /〉〈span class="detail_caption"〉Record origin:〈/span〉 〈a href="http://www.ncbi.nlm.nih.gov/pubmed/23812707" target="_blank"〉PubMed〈/a〉
    Keywords: Escherichia coli/*enzymology ; Guanosine Triphosphate/*chemistry ; Peptide Elongation Factor G/*chemistry ; *Protein Biosynthesis ; Ribosome Subunits, Large, Bacterial/*chemistry ; Ribosomes/*chemistry ; Thermus thermophilus/*enzymology
    Print ISSN: 0036-8075
    Electronic ISSN: 1095-9203
    Topics: Biology , Chemistry and Pharmacology , Computer Science , Medicine , Natural Sciences in General , Physics
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  • 4
    Publication Date: 2014-09-12
    Description: The ribosome is a molecular machine responsible for protein synthesis and a major target for small-molecule inhibitors. Compared to the wealth of structural information available on ribosome-targeting antibiotics in bacteria, our understanding of the binding mode of ribosome inhibitors in eukaryotes is currently limited. Here we used X-ray crystallography to determine 16 high-resolution structures of 80S ribosomes from Saccharomyces cerevisiae in complexes with 12 eukaryote-specific and 4 broad-spectrum inhibitors. All inhibitors were found associated with messenger RNA and transfer RNA binding sites. In combination with kinetic experiments, the structures suggest a model for the action of cycloheximide and lactimidomycin, which explains why lactimidomycin, the larger compound, specifically targets the first elongation cycle. The study defines common principles of targeting and resistance, provides insights into translation inhibitor mode of action and reveals the structural determinants responsible for species selectivity which could guide future drug development.〈br /〉〈span class="detail_caption"〉Notes: 〈/span〉Garreau de Loubresse, Nicolas -- Prokhorova, Irina -- Holtkamp, Wolf -- Rodnina, Marina V -- Yusupova, Gulnara -- Yusupov, Marat -- 294312/European Research Council/International -- England -- Nature. 2014 Sep 25;513(7519):517-22. doi: 10.1038/nature13737. Epub 2014 Sep 10.〈br /〉〈span class="detail_caption"〉Author address: 〈/span〉Institut de Genetique et de Biologie Moleculaire et Cellulaire (IGBMC), INSERM U964, CNRS UMR7104, Universite de Strasbourg, 67404, Illkirch, France. ; Department of Physical Biochemistry, Max Planck Institute for Biophysical Chemistry, Am Fassberg 11, 37077 Gottingen, Germany.〈br /〉〈span class="detail_caption"〉Record origin:〈/span〉 〈a href="http://www.ncbi.nlm.nih.gov/pubmed/25209664" target="_blank"〉PubMed〈/a〉
    Keywords: Base Sequence ; Binding Sites/drug effects ; Crystallography, X-Ray ; Cycloheximide/pharmacology ; Drug Resistance/drug effects ; Eukaryotic Cells/*chemistry/drug effects/enzymology ; Kinetics ; Macrolides/pharmacology ; Models, Molecular ; Molecular Targeted Therapy ; Molecular Weight ; Peptide Chain Elongation, Translational/drug effects ; Peptidyl Transferases/chemistry/metabolism ; Piperidones/pharmacology ; Protein Synthesis Inhibitors/*chemistry/*pharmacology ; RNA, Messenger/genetics/metabolism ; RNA, Transfer/genetics/metabolism ; Ribosome Subunits, Large, Eukaryotic/chemistry/drug effects/metabolism ; Ribosomes/*chemistry/*drug effects/metabolism ; Saccharomyces cerevisiae/*chemistry ; Species Specificity ; Substrate Specificity
    Print ISSN: 0028-0836
    Electronic ISSN: 1476-4687
    Topics: Biology , Chemistry and Pharmacology , Medicine , Natural Sciences in General , Physics
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  • 5
    Publication Date: 2011-08-02
    Description: The active site of the ribosome, the peptidyl transferase centre, catalyses two reactions, namely, peptide bond formation between peptidyl-tRNA and aminoacyl-tRNA as well as the release-factor-dependent hydrolysis of peptidyl-tRNA. Unlike peptide bond formation, peptide release is strongly impaired by mutations of nucleotides within the active site, in particular by base exchanges at position A2602 (refs 1, 2). The 2'-OH group of A76 of the peptidyl-tRNA substrate seems to have a key role in peptide release. According to computational analysis, the 2'-OH may take part in a concerted 'proton shuttle' by which the leaving group is protonated, in analogy to similar current models of peptide bond formation. Here we report kinetic solvent isotope effects and proton inventories (reaction rates measured in buffers with increasing content of deuterated water, D(2)O) of the two reactions catalysed by the active site of the Escherichia coli ribosome. The transition state of the release factor 2 (RF2)-dependent hydrolysis reaction is characterized by the rate-limiting formation of a single strong hydrogen bond. This finding argues against a concerted proton shuttle in the transition state of the hydrolysis reaction. In comparison, the proton inventory for peptide bond formation indicates the rate-limiting formation of three hydrogen bonds with about equal contributions, consistent with a concerted eight-membered proton shuttle in the transition state. Thus, the ribosome supports different rate-limiting transition states for the two reactions that take place in the peptidyl transferase centre.〈br /〉〈span class="detail_caption"〉Notes: 〈/span〉Kuhlenkoetter, Stephan -- Wintermeyer, Wolfgang -- Rodnina, Marina V -- England -- Nature. 2011 Jul 31;476(7360):351-4. doi: 10.1038/nature10247.〈br /〉〈span class="detail_caption"〉Author address: 〈/span〉Max Planck Institute for Biophysical Chemistry, Am Fassberg 11, 37077 Gottingen, Germany.〈br /〉〈span class="detail_caption"〉Record origin:〈/span〉 〈a href="http://www.ncbi.nlm.nih.gov/pubmed/21804565" target="_blank"〉PubMed〈/a〉
    Keywords: Biocatalysis/drug effects ; *Catalytic Domain ; Escherichia coli/chemistry/enzymology ; Escherichia coli Proteins/metabolism ; Hydrogen Bonding ; Hydrogen-Ion Concentration ; Hydrolysis/drug effects ; Kinetics ; Peptide Termination Factors/metabolism ; Protons ; Puromycin/pharmacology ; RNA, Transfer, Met/metabolism ; Ribosomes/*chemistry/drug effects/*metabolism ; Solvents/chemistry ; Substrate Specificity
    Print ISSN: 0028-0836
    Electronic ISSN: 1476-4687
    Topics: Biology , Chemistry and Pharmacology , Medicine , Natural Sciences in General , Physics
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  • 6
    Publication Date: 2015-02-25
    Description: Single particle electron cryomicroscopy (cryo-EM) has recently made significant progress in high-resolution structure determination of macromolecular complexes due to improvements in electron microscopic instrumentation and computational image analysis. However, cryo-EM structures can be highly non-uniform in local resolution and all structures available to date have been limited to resolutions above 3 A. Here we present the cryo-EM structure of the 70S ribosome from Escherichia coli in complex with elongation factor Tu, aminoacyl-tRNA and the antibiotic kirromycin at 2.65-2.9 A resolution using spherical aberration (Cs)-corrected cryo-EM. Overall, the cryo-EM reconstruction at 2.9 A resolution is comparable to the best-resolved X-ray structure of the E. coli 70S ribosome (2.8 A), but provides more detailed information (2.65 A) at the functionally important ribosomal core. The cryo-EM map elucidates for the first time the structure of all 35 rRNA modifications in the bacterial ribosome, explaining their roles in fine-tuning ribosome structure and function and modulating the action of antibiotics. We also obtained atomic models for flexible parts of the ribosome such as ribosomal proteins L9 and L31. The refined cryo-EM-based model presents the currently most complete high-resolution structure of the E. coli ribosome, which demonstrates the power of cryo-EM in structure determination of large and dynamic macromolecular complexes.〈br /〉〈span class="detail_caption"〉Notes: 〈/span〉Fischer, Niels -- Neumann, Piotr -- Konevega, Andrey L -- Bock, Lars V -- Ficner, Ralf -- Rodnina, Marina V -- Stark, Holger -- England -- Nature. 2015 Apr 23;520(7548):567-70. doi: 10.1038/nature14275. Epub 2015 Feb 23.〈br /〉〈span class="detail_caption"〉Author address: 〈/span〉3D Electron Cryomicroscopy Group, Max-Planck-Institute for Biophysical Chemistry, Am Fassberg 11, 37077 Gottingen, Germany. ; Abteilung Molekulare Strukturbiologie, Institut fur Mikrobiologie und Genetik, GZMB, Georg-August Universitat Gottingen, Justus-von Liebig Weg 11, 37077 Gottingen, Germany. ; 1] Molecular and Radiation Biophysics Department, B.P. Konstantinov Petersburg Nuclear Physics Institute of National Research Centre 'Kurchatov Institute', 188300 Gatchina, Russia [2] St Petersburg Polytechnic University, Polytechnicheskaya, 29, 195251 St Petersburg, Russia [3] Department of Physical Biochemistry, Max Planck Institute for Biophysical Chemistry, Am Fassberg 11, 37077 Gottingen, Germany. ; Department of Theoretical and Computational Biophysics, Max Planck Institute for Biophysical Chemistry, Am Fassberg 11, 37077 Gottingen, Germany. ; Department of Physical Biochemistry, Max Planck Institute for Biophysical Chemistry, Am Fassberg 11, 37077 Gottingen, Germany. ; 1] 3D Electron Cryomicroscopy Group, Max-Planck-Institute for Biophysical Chemistry, Am Fassberg 11, 37077 Gottingen, Germany [2] Department of 3D Electron Cryomicroscopy, Institute of Microbiology and Genetics, Georg-August Universitat, 37077 Gottingen, Germany.〈br /〉〈span class="detail_caption"〉Record origin:〈/span〉 〈a href="http://www.ncbi.nlm.nih.gov/pubmed/25707802" target="_blank"〉PubMed〈/a〉
    Keywords: Anti-Bacterial Agents/chemistry/metabolism ; *Cryoelectron Microscopy/methods ; Escherichia coli/*chemistry/*ultrastructure ; Ligands ; Models, Molecular ; Peptide Elongation Factor Tu/*chemistry/metabolism/*ultrastructure ; Pyridones/chemistry/metabolism ; RNA, Bacterial/chemistry/metabolism/ultrastructure ; RNA, Ribosomal/chemistry/metabolism/ultrastructure ; RNA, Transfer/chemistry/metabolism/ultrastructure ; Ribosomes/*chemistry/metabolism/*ultrastructure
    Print ISSN: 0028-0836
    Electronic ISSN: 1476-4687
    Topics: Biology , Chemistry and Pharmacology , Medicine , Natural Sciences in General , Physics
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  • 7
    Publication Date: 2015-11-28
    Description: Protein domains can fold into stable tertiary structures while they are synthesized on the ribosome. We used a high-performance, reconstituted in vitro translation system to investigate the folding of a small five-helix protein domain-the N-terminal domain of Escherichia coli N5-glutamine methyltransferase HemK-in real time. Our observations show that cotranslational folding of the protein, which folds autonomously and rapidly in solution, proceeds through a compact, non-native conformation that forms within the peptide tunnel of the ribosome. The compact state rearranges into a native-like structure immediately after the full domain sequence has emerged from the ribosome. Both folding transitions are rate-limited by translation, allowing for quasi-equilibrium sampling of the conformational space restricted by the ribosome. Cotranslational folding may be typical of small, intrinsically rapidly folding protein domains.〈br /〉〈span class="detail_caption"〉Notes: 〈/span〉Holtkamp, Wolf -- Kokic, Goran -- Jager, Marcus -- Mittelstaet, Joerg -- Komar, Anton A -- Rodnina, Marina V -- New York, N.Y. -- Science. 2015 Nov 27;350(6264):1104-7. doi: 10.1126/science.aad0344.〈br /〉〈span class="detail_caption"〉Author address: 〈/span〉Department of Physical Biochemistry, Max Planck Institute for Biophysical Chemistry, 37077 Gottingen, Germany. ; Center for Gene Regulation in Health and Disease and Department of Biological, Geological and Environmental Sciences, Cleveland State University, Cleveland, OH 44115, USA. ; Department of Physical Biochemistry, Max Planck Institute for Biophysical Chemistry, 37077 Gottingen, Germany. rodnina@mpibpc.mpg.de.〈br /〉〈span class="detail_caption"〉Record origin:〈/span〉 〈a href="http://www.ncbi.nlm.nih.gov/pubmed/26612953" target="_blank"〉PubMed〈/a〉
    Keywords: Escherichia coli Proteins/biosynthesis/chemistry ; Fluorescence Resonance Energy Transfer/*methods ; Peptides/chemistry ; *Protein Biosynthesis ; *Protein Folding ; Protein Methyltransferases/biosynthesis/chemistry ; Protein Structure, Secondary ; Protein Structure, Tertiary ; Proteolysis ; Ribosomes/chemistry/*metabolism ; Time Factors
    Print ISSN: 0036-8075
    Electronic ISSN: 1095-9203
    Topics: Biology , Chemistry and Pharmacology , Computer Science , Medicine , Natural Sciences in General , Physics
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  • 8
  • 9
    Publication Date: 1995-03-14
    Print ISSN: 0027-8424
    Electronic ISSN: 1091-6490
    Topics: Biology , Medicine , Natural Sciences in General
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  • 10
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