The Ribosomal Peptidyl Transferase Center: Structure, Function, Evolution, Inhibition
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Evolution of Translation EF-Tu: Trna
University of Illinois at Urbana-Champaign Luthey-Schulten Group NIH Resource for Macromolecular Modeling and Bioinformatics Computational Biophysics Workshop Evolution of Translation EF-Tu: tRNA VMD Developer: John Stone MultiSeq Developers Tutorial Authors Elijah Roberts Ke Chen John Eargle John Eargle Dan Wright Zhaleh Ghaemi Jonathan Lai Zan Luthey-Schulten August 2014 A current version of this tutorial is available at http://www.scs.illinois.edu/~schulten/tutorials/ef-tu CONTENTS 2 Contents 1 Introduction 3 1.1 The Elongation Factor Tu . 3 1.2 Getting Started . 4 1.2.1 Requirements . 4 1.2.2 Copying the tutorial files . 4 1.2.3 Working directory . 4 1.2.4 Preferences . 4 1.3 Configuring BLAST for MultiSeq . 5 2 Comparative Analysis of EF-Tu 5 2.1 Finding archaeal EF1A sequences . 6 2.2 Aligning archaeal sequences and removing redundancy . 8 2.3 Finding bacteria EF-Tu sequences . 11 2.4 Performing ClustalW Multiple Sequence and Profile-Profile Align- ments . 12 2.5 Creating Multiple Sequence with MAFFT . 16 2.6 Conservation of EF-Tu among the Bacteria . 16 2.7 Finding conserved residues across the bacterial and archaeal do- mains . 20 2.8 EF-Tu Interface with the Ribosome . 21 3 Computing a Maximum Likelihood Phylogenetic Tree with RAxML 23 3.1 Load the Phylogenetic Tree into MultiSeq . 25 3.2 Reroot and Manipulate the Phylogenetic Tree . 25 4 MultiSeq TCL Scripting: Genomic Context 27 5 Appendix A 30 5.1 Building a BLAST Database . 30 6 Appendix B 31 6.1 Saving QR subset of alignments in PHYLIP and FASTA format 31 6.2 Calculating Maximum Likelihood Trees with RAxML . -
Crystal Structure of the Eukaryotic 60S Ribosomal Subunit in Complex with Initiation Factor 6
Research Collection Doctoral Thesis Crystal structure of the eukaryotic 60S ribosomal subunit in complex with initiation factor 6 Author(s): Voigts-Hoffmann, Felix Publication Date: 2012 Permanent Link: https://doi.org/10.3929/ethz-a-007303759 Rights / License: In Copyright - Non-Commercial Use Permitted This page was generated automatically upon download from the ETH Zurich Research Collection. For more information please consult the Terms of use. ETH Library ETH Zurich Dissertation No. 20189 Crystal Structure of the Eukaryotic 60S Ribosomal Subunit in Complex with Initiation Factor 6 A dissertation submitted to ETH ZÜRICH for the degree of Doctor of Sciences (Dr. sc. ETH Zurich) presented by Felix Voigts-Hoffmann MSc Molecular Biotechnology, Universität Heidelberg born April 11, 1981 citizen of Göttingen, Germany accepted on recommendation of Prof. Dr. Nenad Ban (Examiner) Prof. Dr. Raimund Dutzler (Co-examiner) Prof. Dr. Rudolf Glockshuber (Co-examiner) 2012 blank page ii Summary Ribosomes are large complexes of several ribosomal RNAs and dozens of proteins, which catalyze the synthesis of proteins according to the sequence encoded in messenger RNA. Over the last decade, prokaryotic ribosome structures have provided the basis for a mechanistic understanding of protein synthesis. While the core functional centers are conserved in all kingdoms, eukaryotic ribosomes are much larger than archaeal or bacterial ribosomes. Eukaryotic ribosomal rRNA and proteins contain extensions or insertions to the prokaryotic core, and many eukaryotic proteins do not have prokaryotic counterparts. Furthermore, translation regulation and ribosome biogenesis is much more complex in eukaryotes, and defects in components of the translation machinery are associated with human diseases and cancer. -
Syntheses and Biological Activity of Sparsomycin and Analogs
SYNTHESES AND BIOLOGICAL ACTIVITY OF SPARSOMYCIN AND ANALOGS THE CHEMISTRY OF CHIRAL FUNCTIONALIZED SULFOXIDES AND SULTINES DERIVED FROM CYSTEINE ROB M.J. LISKAMP SYNTHESES AND BIOLOGICAL ACTIVITY OF SPARSOMYCIN AND ANALOGS THE CHEMISTRY OF CHIRAL FUNCTIONALIZED SULFOXIDES AND SULTINES DERIVED FROM CYSTEINE Promotor : Prof. Dr. R.J.F. Nivard Co-referent : Dr. H.C.J. Ottenheijm SYNTHESES AND BIOLOGICAL ACTIVITY OF SPARSOMYCIN AND ANALOGS THE CHEMISTRY OF CHIRAL FUNCTIONALIZED SULFOXIDES AND SULTINES DERIVED FROM CYSTEINE PROEFSCHRIFT TER VERKRIJGING VAN DE GRAAD VAN DOCTOR IN DE WISKUNDE EN NATUURWETENSCHAPPEN AAN DE KATHOLIEKE UNIVERSITEIT VAN NIJMEGEN OP GEZAG VAN DE RECTOR MAGNIFICUS PROF. DR. J.H.G.I. GIESBERS VOLGENS HET BESLUIT VAN HET COLLEGE VAN DEKANEN IN HET OPENBAAR TE VERDEDIGEN OP VRIJDAG 10 DECEMBER 1982 DES NAMIDDAGS OM 4 UUR DOOR ROBERTUS MATTHIAS JOSEPH LISKAMP GEBOREN TE NIJMEGEN NIJMEGEN 1982 Dit proefschrift vormt de neerslag van vier jaar wetenschappelijk onderzoek. Ik wil iedereen - en dat waren er zeer velen - bedanken, die hieraan gedurende die tijd door hun stimulerende discussie of door de feitelijke uitvoering deel hadden. Zij gaven mede richting en vorm, niet alleen organisch maar ook biochemisch/biologisch en fysisch chemisch, aan het verrichte onderzoek. Daarnaast wil ik hen bedanken die een bijdrage geleverd hebben aan de onmis bare technische uitvoering van het in dit proefschrift beschreven onderzoek. En tenslotte wil ik ook hen bedanken die aan de vormgeving van het proef schrift hun medewerking hebben verleend. Tekeningen : Wim van Luijn en de afdeling illustratie Type-werk : Dorine Hesselink-Balvert Afbeelding op de omslag: Een 'stacked plot' van een 2D-spin-echo correlated spectrum van synthetisch sparsomycine verzorgd door Wim Guijt This research was supported by the Netherlands Foundation for Chemical Research (SON) with financial aid from the Netherlands Organization for the Advancement of Pure Research (ZWO). -
Effects of Oxidative Stress on Protein Translation
International Journal of Molecular Sciences Review Effects of Oxidative Stress on Protein Translation: Implications for Cardiovascular Diseases Arnab Ghosh * and Natalia Shcherbik * Department for Cell Biology and Neuroscience, School of Osteopathic Medicine, Rowan University, 2 Medical Center Drive, Stratford, NJ 08084, USA * Correspondence: [email protected] (A.G.); [email protected] (N.S.); Tel.: +1-856-566-6907 (A.G.); +1-856-566-6914 (N.S.) Received: 24 March 2020; Accepted: 9 April 2020; Published: 11 April 2020 Abstract: Cardiovascular diseases (CVDs) are a group of disorders that affect the heart and blood vessels. Due to their multifactorial nature and wide variation, CVDs are the leading cause of death worldwide. Understanding the molecular alterations leading to the development of heart and vessel pathologies is crucial for successfully treating and preventing CVDs. One of the causative factors of CVD etiology and progression is acute oxidative stress, a toxic condition characterized by elevated intracellular levels of reactive oxygen species (ROS). Left unabated, ROS can damage virtually any cellular component and affect essential biological processes, including protein synthesis. Defective or insufficient protein translation results in production of faulty protein products and disturbances of protein homeostasis, thus promoting pathologies. The relationships between translational dysregulation, ROS, and cardiovascular disorders will be examined in this review. Keywords: protein translation; ribosome; RNA; IRES; uORF; miRNA; cardiovascular diseases; reactive oxygen species; oxidative stress; antioxidants 1. Introduction The process of protein synthesis, or protein translation, constitutes the last and final step of the central dogma of molecular biology: assembly of polypeptides based on the information encoded by mRNAs. This complex process employs multiple essential players, including ribosomes, mRNAs, tRNAs, and numerous translational factors, enzymes, and regulatory proteins. -
Ef-G:Trna Dynamics During the Elongation Cycle of Protein Synthesis
University of Pennsylvania ScholarlyCommons Publicly Accessible Penn Dissertations 2015 Ef-G:trna Dynamics During the Elongation Cycle of Protein Synthesis Rong Shen University of Pennsylvania, [email protected] Follow this and additional works at: https://repository.upenn.edu/edissertations Part of the Biochemistry Commons Recommended Citation Shen, Rong, "Ef-G:trna Dynamics During the Elongation Cycle of Protein Synthesis" (2015). Publicly Accessible Penn Dissertations. 1131. https://repository.upenn.edu/edissertations/1131 This paper is posted at ScholarlyCommons. https://repository.upenn.edu/edissertations/1131 For more information, please contact [email protected]. Ef-G:trna Dynamics During the Elongation Cycle of Protein Synthesis Abstract During polypeptide elongation cycle, prokaryotic elongation factor G (EF-G) catalyzes the coupled translocations on the ribosome of mRNA and A- and P-site bound tRNAs. Continued progress has been achieved in understanding this key process, including results of structural, ensemble kinetic and single- molecule studies. However, most of work has been focused on the pre-equilibrium states of this fast process, leaving the real time dynamics, especially how EF-G interacts with the A-site tRNA in the pretranslocation complex, not fully elucidated. In this thesis, the kinetics of EF-G catalyzed translocation is investigated by both ensemble and single molecule fluorescence resonance energy transfer studies to further explore the underlying mechanism. In the ensemble work, EF-G mutants were designed and expressed successfully. The labeled EF-G mutants show good translocation activity in two different assays. In the smFRET work, by attachment of a fluorescent probe at position 693 on EF-G permits monitoring of FRET efficiencies to sites in both ribosomal protein L11 and A-site tRNA. -
Mitochondrial Translation and Its Impact on Protein Homeostasis And
Mitochondrial translation and its impact on protein homeostasis and aging Tamara Suhm Academic dissertation for the Degree of Doctor of Philosophy in Biochemistry at Stockholm University to be publicly defended on Friday 15 February 2019 at 09.00 in Magnélisalen, Kemiska övningslaboratoriet, Svante Arrhenius väg 16 B. Abstract Besides their famous role as powerhouse of the cell, mitochondria are also involved in many signaling processes and metabolism. Therefore, it is unsurprising that mitochondria are no isolated organelles but are in constant crosstalk with other parts of the cell. Due to the endosymbiotic origin of mitochondria, they still contain their own genome and gene expression machinery. The mitochondrial genome of yeast encodes eight proteins whereof seven are core subunits of the respiratory chain and ATP synthase. These subunits need to be assembled with subunits imported from the cytosol to ensure energy supply of the cell. Hence, coordination, timing and accuracy of mitochondrial gene expression is crucial for cellular energy production and homeostasis. Despite the central role of mitochondrial translation surprisingly little is known about the molecular mechanisms. In this work, I used baker’s yeast Saccharomyces cerevisiae to study different aspects of mitochondrial translation. Exploiting the unique possibility to make directed modifications in the mitochondrial genome of yeast, I established a mitochondrial encoded GFP reporter. This reporter allows monitoring of mitochondrial translation with different detection methods and enables more detailed studies focusing on timing and regulation of mitochondrial translation. Furthermore, employing insights gained from bacterial translation, we showed that mitochondrial translation efficiency directly impacts on protein homeostasis of the cytoplasm and lifespan by affecting stress handling. -
Peptidyl-Transferase Ribozymes: Trans Reactions, Structural Characterization and Ribosomal RNA-Like Features Wang Zhang* and Thomas R Cech
Research Paper 539 Peptidyl-transferase ribozymes: trans reactions, structural characterization and ribosomal RNA-like features Wang Zhang* and Thomas R Cech Background: One of the most significant questions in understanding the origin Address: Howard Hughes Medical Institute, of life concerns the order of appearance of DNA, RNA and protein during early Department of Chemistry and Biochemistry, University of Colorado, Boulder, CO 80309-0215, biological evolution. If an ‘RNA world’ was a precursor to extant life, RNA must USA. be able not only to catalyze RNA replication but also to direct peptide synthesis. Iterative RNA selection previously identified catalytic RNAs (ribozymes) that form *Present address: Program in Molecular Medicine, amide bonds between RNA and an amino acid or between two amino acids. University of Massachusetts Medical Center, 373 Plantation Street, Worcester, MA 01605, USA. Results: We characterized peptidyl-transferase reactions catalyzed by two Correspondence: Thomas R Cech different families of ribozymes that use substrates that mimic A site and P site E-mail: [email protected] tRNAs. The family II ribozyme secondary structure was modeled using chemical Key words: metal ions, peptidyl transferase, modification, enzymatic digestion and mutational analysis. Two regions ribosomal RNA structure, RNA catalysis, RNA resemble the peptidyl-transferase region of 23s ribosomal RNA in sequence structure and structural context; these regions are important for peptide-bond formation. A shortened form of this ribozyme was engineered to catalyze intermolecular Received: 3 August 1998 (‘trans’) peptide-bond formation, with the two amino-acid substrates binding Revisions requested: 18 August 1998 Revisions received: 26 August 1998 through an attached AMP or oligonucleotide moiety. -
ANA-1 Murine Macrophages Ribosomal Peptidyl Transferase Activity in Cleavage Is Associated with Inhibition of Nitric Oxide-Depen
Nitric Oxide-Dependent Ribosomal RNA Cleavage Is Associated with Inhibition of Ribosomal Peptidyl Transferase Activity in ANA-1 Murine Macrophages This information is current as of October 2, 2021. Charles Q. Cai, Hongtao Guo, Rebecca A. Schroeder, Cecile Punzalan and Paul C. Kuo J Immunol 2000; 165:3978-3984; ; doi: 10.4049/jimmunol.165.7.3978 http://www.jimmunol.org/content/165/7/3978 Downloaded from References This article cites 13 articles, 5 of which you can access for free at: http://www.jimmunol.org/content/165/7/3978.full#ref-list-1 http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication by guest on October 2, 2021 *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2000 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Nitric Oxide-Dependent Ribosomal RNA Cleavage Is Associated with Inhibition of Ribosomal Peptidyl Transferase Activity in ANA-1 Murine Macrophages1 Charles Q. Cai,* Hongtao Guo,* Rebecca A. Schroeder,† Cecile Punzalan,* Paul C. Kuo2* NO can regulate specific cellular functions by altering transcriptional programs and protein reactivity. -
Mutations in C12orf65 in Patients with Encephalomyopathy and a Mitochondrial Translation Defect
View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector REPORT Mutations in C12orf65 in Patients with Encephalomyopathy and a Mitochondrial Translation Defect Hana Antonicka,1,7 Elsebet Østergaard,2,7 Florin Sasarman,1 Woranontee Weraarpachai,1 Flemming Wibrand,2 Anne Marie B. Pedersen,3 Richard J. Rodenburg,4 Marjo S. van der Knaap,5 Jan A.M. Smeitink,4 Zofia M. Chrzanowska-Lightowlers,6 and Eric A. Shoubridge1,* We investigated the genetic basis for a global and uniform decrease in mitochondrial translation in fibroblasts from patients in two unre- lated pedigrees who developed Leigh syndrome, optic atrophy, and ophthalmoplegia. Analysis of the assembly of the oxidative phos- phorylation complexes showed severe decreases of complexes I, IV, and V and a smaller decrease in complex III. The steady-state levels of mitochondrial mRNAs, tRNAs, and rRNAs were not reduced, nor were those of the mitochondrial translation elongation factors or the protein components of the mitochondrial ribosome. Using homozygosity mapping, we identified a 1 bp deletion in C12orf65 in one patient, and DNA sequence analysis showed a different 1 bp deletion in the second patient. Both mutations predict the same premature stop codon. C12orf65 belongs to a family of four mitochondrial class I peptide release factors, which also includes mtRF1a, mtRF1, and Ict1, all characterized by the presence of a GGQ motif at the active site. However, C12orf65 does not exhibit peptidyl-tRNA hydrolase activity in an in vitro assay with bacterial ribosomes. We suggest that it might play a role in recycling abortive peptidyl-tRNA species, released from the ribosome during the elongation phase of translation. -
Inhibitory Effect of Ef G and Gmppcp on Peptidyl Transferase
View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector Volume 44, number 3 FEBS LETTERS August 1974 INHIBITORY EFFECT OF EF G AND GMPPCP ON PEPTIDYL TRANSFERASE Tadahiko OTAKA and Akira KAJI Department of Microbiology, School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19174, USA Received 13 May 1974 1. Introduct;on from Miles Laboratories. Fusidic acid (Na salt) (Batch # 14) was a gift from E. R. Squibb and Sons Co. Evidence has been accumulating which suggests that through the courtesy of Dr. A. Laskin. The specific the binding of EF G and GTP takes place at or near activities of [“Cl phenylalanine and [“HI methionine the A site of the ribosome which accepts the complex were 455 Ci/mole and 3270 Ci/mole, respectively. of EF Tu, aminoacyl-tRNA and GTP. Thus, the com- Preparation of [I4 C] phenylalanyl-tRNA [7] , N- plex of GMPPCP with EF G as well as fusidic acid and acetyl [‘4C]phenylanyl-tRNA 181 and formyl [3 HI- GDP with EF G has been found to inhibit the binding methionyl-tRNA [9] were obtained as described of aminoacyl-tRNA effectively at the A site [l-4] . previously. The counting efficiencies of I4 C and 3 H These results suggested that the site for EF G and were lo6 cpm/pc and 4.9 X 10’ cpm/gc, respect- GMPPCP binding may be identical to or overlap with ively. that for EF G, GDP, and fusidic acid. On the other hand, recent evidence suggests that these sites are 2.2. -
Induced-Fit of the Peptidyl-Transferase Center of the Ribosome and Conformational Freedom of the Esterified Amino Acids
bioRxiv preprint doi: https://doi.org/10.1101/032516; this version posted December 2, 2015. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Induced-fit of the peptidyl-transferase center of the ribosome and conformational freedom of the esterified amino acids Jean Lehmann1 Institute for Integrative Biology of the Cell (I2BC), CEA, CNRS, Université Paris-Sud, Campus Paris-Saclay, 91198 Gif-sur-Yvette, France 1Email: [email protected] Running title: Induced-fit of the PTC and aminoacyl esters. Keywords: Ribosome, peptidyl-transferase center, induced-fit, aminoacyl-tRNA. ABSTRACT The catalytic site of most enzymes can essentially deal with only one substrate. In contrast, the ribosome is capable of polymerizing at a similar rate at least 20 different kinds of amino acids from aminoacyl-tRNA carriers while using just one catalytic site, the peptidyl-transferase center (PTC). An induced-fit mechanism has been uncovered in the PTC, but a possible connection between this mechanism and the uniform handling of the substrates has not been investigated. We present an analysis of published ribosome structures supporting the hypothesis that the induced- fit eliminates unreactive rotamers predominantly populated for some A-site aminoacyl esters before induction. We show that this hypothesis is fully consistent with the wealth of kinetic data obtained with these substrates. Our analysis reveals that induction constrains the amino acids into a reactive conformation in a side-chain independent manner. -
Sparsomycin and Its Analogues
PDF hosted at the Radboud Repository of the Radboud University Nijmegen The following full text is a publisher's version. For additional information about this publication click this link. http://hdl.handle.net/2066/113516 Please be advised that this information was generated on 2021-10-06 and may be subject to change. SPARSOMYCIN AND ITS ANALOGUES A preclinical study on novel anticancer drugs Promotores: Prof. Dr. D.J.Th. Wagener Prof. Dr. E. van der Kleijn Prof. Dr. H.C.J. Ottenheijm (V.U., Amsterdam) SPARSOMYCIN AND ITS ANALOGUES A preclinical study on novel anticancer drugs een wetenschappelijke proeve op het gebied van de geneeskunde en tandheelkunde PROEFSCHRIFT ter verkrijging van de graad van doctor aan de Katholieke Universiteit te Nijmegen, volgens besluit van het college van decanen in het openbaar te verdedigen op dinsdag 7 juni 1988 des namiddags te 3.30 uur door Zbigniew Zylicz geboren op 13 juni 1955 te Wroclaw (Polen) Nijmegen, 1988 druk: reprografie faculteit der geneeskunde en tandheelkunde The investigations have been performed at the Department of Internal Medicine, Division of Medical Oncology, and the Department of Clinical Pharmacy, University Hospital Nijmegen, The Netherlands. These studies were supported by The Netherlands Cancer Foundation Queen Wilhelmina Fund (grant nr SNUKC-84-1) and Pharmachemie BV, Haarlem, The Netherlands. Pharmachemie BV, Haarlem, is also acknowledged for financial support of publication of this thesis. Yr3/V)ornm vr? тле/ г$и Abbreviat ions AUG - area under the plasma concentration versus time curve ARA-C - cytosine arabinoside CHO - Chinese hamster ovary cells CL-j, - creatinine clearance CL™ - non-renal clearance CLp - renal clearance С - steady state concentration D - dose dSm - deshydroxy-sparsomycin EdSm - ethyl-deshydroxy-sparsomycin 5-FU - 5-fluorouracil i.d.