DOCSLIB.ORG

Ef-G:Trna Dynamics During the Elongation Cycle of Protein Synthesis

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
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. The EF-G:L11 FRET efficiency is constant during the entire EF-G occupancy on the ribosome, while the EF-G:tRNA FRET proceeds from high to intermediate to low FRET values. Transition from intermediate to low is the rate-limiting step with a small FRET change, whereas transition from high to intermediate is fast, showing a large FRET change. In total, we capture a ~12 Ã? relative movement of A-site tRNA away from EF-G during translocation. This indicates that EF-G:A-site tRNA distance increases progressively during translocation, with our results providing the first good estimates of the timing of such changes. This EF-G:tRNA smFRET measurement was applied to bifunctional rhodamine labeled EF-G derivatives (BR-EF-G) and comparable kinetic results were obtained. FRET changes of BR-EF-G:Cy5-tRNA pair are larger if the BR group is located closer to the EF-G hinge region, suggesting that parts of domain IV of EF-G, which penetrate to the decoding region of the ribosome, move together with the A-site tRNA during translocation. Degree Type Dissertation Degree Name Doctor of Philosophy (PhD) Graduate Group Chemistry First Advisor Barry S. Cooperman Keywords EF-G, SmFRET, tRNA, ribosome, kinetics Subject Categories Biochemistry This dissertation is available at ScholarlyCommons: https://repository.upenn.edu/edissertations/1131 EF-G:tRNA DYNAMICS DURING THE ELONGATION CYCLE OF PROTEIN SYNTHESIS Rong Shen A DISSERTATION in Chemistry Presented to the Faculties of the University of Pennsylvania in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy 2015 Supervisor of Dissertation Dr. Barry S. Cooperman, Professor of Chemistry Graduate Group Chairperson Dr. Gary A. Molander, Hirschmann-Makineni Professor of Chemistry Dissertation Committee: Dr. Ivan J. Dmochowski, Associate Professor of Chemistry Dr. Yale E. Goldman, Professor of Physiology Dr. E. James Petersson, Assistant Professor of Chemistry Dedicated to my father Changming Shen and my mother Yan Shen ii ACKNOWLEDGEMENT A PhD journey is never easy. It is a pleasure and an honor to thank many people who made this thesis possible. First and foremost, this thesis would never have happened without the encouragement, guidance and support from my research advisor Dr. Barry S. Cooperman. It is my highest honor to be one of his students. From him, I have learned how to think critically which would greatly benefit my future career. His enthusiasm toward science and immense knowledge not only inspire me but also make the learning process a great fun for me. After five years research work in Dr. Cooperman’s lab, I find where my interest is and how I should pursue my future career. I would like to show my greatest gratitude towards our major collaborator with this project. Dr. Yale Goldman is not only a committee member, but also a very close collaborator with our group. In the text you will notice that a large portion of my work is smFRET study which was carried out in Dr. Yale Goldman’s lab. I attended his journal club and enjoy discussing my data with him. His inspiring insights into my work have proved to be priceless. I am indebted to my dissertation committee members, Prof. Ivan Dmochowski and Prof. Petersson for their constructive suggestions to my work and support to my study at Penn. I learned how to think about my research from other perspectives and without their help, it would have been impossible for me to accomplish my thesis. It is a great pleasure for me to thank all my current and past group members. When I first joined in the lab, Dr. Haibo Zhang taught me how to purify protein and helped iii me with my everyday work. Dr. Hanqing Liu helped me with the quench flow experiment. I learned how to do kinetic experiments and analyze the complicated data with the help of Dr. Wei Liu, who also shared a lot of invaluable experience with me about data interpretation. Dr. Ian Farrell and Dr. Jaskiran Kaur helped me with tRNA preparation. Dr. Chunlai Chen and Dr. Darius Kavaliauskas helped me with the smFRET experiment and spent a lot of time on discussing smFRET data with me. Dr. Amy Weil, Dr. Dulce Alonso, Dr. Gabi Rosenblum, Mr. Yuanwei Chen, Mr. Martin Ng and Ms. Adrienne Pesce have been incredible to work with and learn from. I would like to thank my roommates Na Zhang and Dr. Zhaoxia Qian, my dear friends Dr. Han Wei, Dr. Jiadi Zhang and Yanran Ai, with whom I came to Penn six years ago and Jie Qing, Fang Liu, Minyan Li, with whom I shared laughter and interesting conversations. I own my deepest gratitude to my family. I would like to thank my parents for their love, support and encouragement during these years and I will never know how far I could have made it without their support. I also want to thank my dog, lele, who is one of my dear family members and brought a lot of laughter to me. iv ABSTRACT EF-G:tRNA DYNAMICS DURING THE ELONGATION CYCLE OF PROTEIN SYNTHESIS Rong Shen Barry S. Cooperman 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. The EF-G:L11 FRET efficiency is constant v during the entire EF-G occupancy on the ribosome, while the EF-G:tRNA FRET proceeds from high to intermediate to low FRET values. Transition from intermediate to low is the rate-limiting step with a small FRET change, whereas transition from high to intermediate is fast, showing a large FRET change. In total, we capture a ~12 Å relative movement of A-site tRNA away from EF-G during translocation. This indicates that EF-G:A-site tRNA distance increases progressively during translocation, with our results providing the first good estimates of the timing of such changes. This EF- G:tRNA smFRET measurement was applied to bifunctional rhodamine labeled EF-G derivatives (BR-EF-G) and comparable kinetic results were obtained. FRET changes of BR-EF-G:Cy5-tRNA pair are larger if the BR group is located closer to the EF-G hinge region, suggesting that parts of domain IV of EF-G, which penetrate to the decoding region of the ribosome, move together with the A-site tRNA during translocation. vi TABLE OF CONTENTS ACKNOWLEDGEMENT ........................................................................................................... iii ABSTRACT ................................................................................................................................... v LIST OF ILLUSTRATIONS ..................................................................................................... xiii CHAPTER I INTRODUCTION .................................................................................................. 1 1.1 The central dogma and ribosome-catalyzed translation .................................................
Load more

Recommended publications
  • 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 .
    [Show full text]
  • Crystal Structure of a Translation Termination Complex Formed with Release Factor RF2
    Crystal structure of a translation termination complex formed with release factor RF2 Andrei Korosteleva,b,1, Haruichi Asaharaa,b,1,2, Laura Lancastera,b,1, Martin Laurberga,b, Alexander Hirschia,b, Jianyu Zhua,b, Sergei Trakhanova,b, William G. Scotta,c, and Harry F. Nollera,b,3 aCenter for Molecular Biology of RNA and Departments of bMolecular, Cell and Developmental Biology and cChemistry and Biochemistry, University of California, Santa Cruz, CA 95064 Contributed by Harry F. Noller, October 30, 2008 (sent for review October 22, 2008) We report the crystal structure of a translation termination com- and G530 of 16S rRNA, and recognized separately by interac- plex formed by the Thermus thermophilus 70S ribosome bound tions with Gln-181 and Thr-194. Stop codon recognition by RF1 with release factor RF2, in response to a UAA stop codon, solved also involves a network of interactions with other structural at 3 Å resolution. The backbone of helix ␣5 and the side chain of elements of RF1, including critical main-chain atoms and con- serine of the conserved SPF motif of RF2 recognize U1 and A2 of the served features of 16S rRNA (9). stop codon, respectively. A3 is unstacked from the first 2 bases, Many studies have implicated the conserved GGQ motif in contacting Thr-216 and Val-203 of RF2 and stacking on G530 of 16S domain 3, present in the release factors of all three primary rRNA. The structure of the RF2 complex supports our previous domains of life, in the hydrolysis reaction. Although the side proposal that conformational changes in the ribosome in response chain of the conserved glutamine has been proposed to play a to recognition of the stop codon stabilize rearrangement of the role in catalysis (10, 11), elimination of its side-chain amide switch loop of the release factor, resulting in docking of the group by mutation of this residue to alanine, for example, universally conserved GGQ motif in the PTC of the 50S subunit.
    [Show full text]
  • Effects of Single Amino Acid Deficiency on Mrna Translation Are Markedly
    www.nature.com/scientificreports OPEN Efects of single amino acid defciency on mRNA translation are markedly diferent for methionine Received: 12 December 2016 Accepted: 4 May 2018 versus leucine Published: xx xx xxxx Kevin M. Mazor, Leiming Dong, Yuanhui Mao, Robert V. Swanda, Shu-Bing Qian & Martha H. Stipanuk Although amino acids are known regulators of translation, the unique contributions of specifc amino acids are not well understood. We compared efects of culturing HEK293T cells in medium lacking either leucine, methionine, histidine, or arginine on eIF2 and 4EBP1 phosphorylation and measures of mRNA translation. Methionine starvation caused the most drastic decrease in translation as assessed by polysome formation, ribosome profling, and a measure of protein synthesis (puromycin-labeled polypeptides) but had no signifcant efect on eIF2 phosphorylation, 4EBP1 hyperphosphorylation or 4EBP1 binding to eIF4E. Leucine starvation suppressed polysome formation and was the only tested condition that caused a signifcant decrease in 4EBP1 phosphorylation or increase in 4EBP1 binding to eIF4E, but efects of leucine starvation were not replicated by overexpressing nonphosphorylatable 4EBP1. This suggests the binding of 4EBP1 to eIF4E may not by itself explain the suppression of mRNA translation under conditions of leucine starvation. Ribosome profling suggested that leucine deprivation may primarily inhibit ribosome loading, whereas methionine deprivation may primarily impair start site recognition. These data underscore our lack of a full
    [Show full text]
  • Structural Aspects of Translation Termination on the Ribosome
    View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by eScholarship@UMMS University of Massachusetts Medical School eScholarship@UMMS RNA Therapeutics Institute Publications RNA Therapeutics Institute 2011-08-01 Structural aspects of translation termination on the ribosome Andrei A. Korostelev University of Massachusetts Medical School Let us know how access to this document benefits ou.y Follow this and additional works at: https://escholarship.umassmed.edu/rti_pubs Part of the Biochemistry, Biophysics, and Structural Biology Commons, Cell and Developmental Biology Commons, Genetics and Genomics Commons, and the Therapeutics Commons Repository Citation Korostelev AA. (2011). Structural aspects of translation termination on the ribosome. RNA Therapeutics Institute Publications. https://doi.org/10.1261/rna.2733411. Retrieved from https://escholarship.umassmed.edu/rti_pubs/33 This material is brought to you by eScholarship@UMMS. It has been accepted for inclusion in RNA Therapeutics Institute Publications by an authorized administrator of eScholarship@UMMS. For more information, please contact [email protected]. REVIEW Structural aspects of translation termination on the ribosome ANDREI A. KOROSTELEV1 RNA Therapeutics Institute and Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical School, Worcester, Massachusetts 01605, USA ABSTRACT Translation of genetic information encoded in messenger RNAs into polypeptide sequences is carried out by ribosomes in all organisms. When a full protein is synthesized, a stop codon positioned in the ribosomal A site signals termination of translation and protein release. Translation termination depends on class I release factors. Recently, atomic-resolution crystal structures were determined for bacterial 70S ribosome termination complexes bound with release factors RF1 or RF2.
    [Show full text]
  • Allosteric Collaboration Between Elongation Factor G and the Ribosomal L1 Stalk Directs Trna Movements During Translation
    Allosteric collaboration between elongation factor G and the ribosomal L1 stalk directs tRNA movements during translation Jingyi Feia, Jonathan E. Bronsona, Jake M. Hofmanb,1, Rathi L. Srinivasc, Chris H. Wigginsd and Ruben L. Gonzalez, Jr.a,2 aDepartment of Chemistry, bDepartment of Physics, cThe Fu Foundation School of Engineering and Applied Science and dDepartment of Applied Physics and Applied Mathematics, Columbia University, New York, NY 10027 1Current address: Yahoo! Research, 111 West 40th Street, 17th Floor, NewYork, NY 10018 2To whom correspondence may be addressed. E-mail: [email protected] Classification: Biological Sciences, Biochemistry ABSTRACT Determining the mechanism by which transfer RNAs (tRNAs) rapidly and precisely transit through the ribosomal A, P and E sites during translation remains a major goal in the study of protein synthesis. Here, we report the real-time dynamics of the L1 stalk, a structural element of the large ribosomal subunit that is implicated in directing tRNA movements during translation. Within pre-translocation ribosomal complexes, the L1 stalk exists in a dynamic equilibrium between open and closed conformations. Binding of elongation factor G (EF-G) shifts this equilibrium towards the closed conformation through one of at least two distinct kinetic mechanisms, where the identity of the P-site tRNA dictates the kinetic route that is taken. Within post-translocation complexes, L1 stalk dynamics are dependent on the presence and identity of the E-site tRNA. Collectively, our data demonstrate that EF-G and the L1 stalk allosterically collaborate to direct tRNA translocation from the P to the E sites, and suggest a model for the release of E-site tRNA.
    [Show full text]
  • Detecting Translational Regulation by Change Point Analysis of Ribosome Profiling Datasets
    bioRxiv preprint doi: https://doi.org/10.1101/003210; this version posted March 5, 2014. 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 4.0 International license. Detecting translational regulation by change point analysis of ribosome profiling datasets Zupanic A1, Meplan C2, Grellscheid SN3, Mathers JC1, Kirkwood TBL1, Hesketh JE2, Shanley DP1 1Centre for Integrated Systems Biology of Ageing & Nutrition, Institute for Ageing and Health, Newcastle University, Newcastle-upon-Tyne, NE4 5PL, UK 2Institute for Cell and Molecular Biosciences and Human Nutrition Research Centre, Newcastle University, Newcastle-upon-Tyne, NE2 4HH, UK 3School of Biological and Biomedical Sciences, Durham University, Durham DH1 3LE, UK Running title: Translational regulation in ribosome profiles Keywords: ribosome profiling, translation regulation, change point, mathematical model Corresponding author: Daryl Shanley Centre for Integrated Systems Biology of Ageing & Nutrition, Institute for Ageing and Health, Newcastle University, NE4 5PL, UK Email: [email protected] Fax: +441912481101 1 bioRxiv preprint doi: https://doi.org/10.1101/003210; this version posted March 5, 2014. 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 4.0 International license. Abstract Ribo-Seq maps the location of translating ribosomes on mature mRNA transcripts. While ribosome density is constant along the length of the mRNA coding region, it can be altered by translational regulatory events.
    [Show full text]
  • YEAST CELLS MAY USE AUC OR AAG AS INITIATION CODON for PROTEIN SYNTHESIS by OLE OLSEN
    Carlsberg Res. Commun. Vol. 52, p. 83-90, 1987 YEAST CELLS MAY USE AUC OR AAG AS INITIATION CODON FOR PROTEIN SYNTHESIS by OLE OLSEN Department of Physiology, Carlsberg Laboratory, Gamle Cadsbergvej 10, DK-2500 Copenhagen Valby Keywords: Recombinant DNA, translation initiation, secretion A yeast expression plasmid without an ATG codon for initiation of mouse a-amylase protein synthesis directs the synthesis and secretion of active enzyme indistinguishable from both native mouse a-amylase and amylase synthesized from plasmids with normal AT(3 initiation codons. The initiation of amylase synthesis directed by this plasmid is at either an AUC or an AAG codon. In either case the amino acid sequence of the hydrophobic core and peptidase cleaving region of the signal peptide are normal, and the protein translation remains in frame with the structural gene of the mouse a-amylase. 1. INTRODUCTION mal context for initiation was 6NNAUGGA A where Initiation of protein synthesis in eukaryotes is a purine in position -3 (three nucleotides up- catalysed by a relatively large number of specific stream of the AUG initiator codon) is most eukaryotic initation factors (eIFs) bringing highly conserved. about the formation of the 80S initiation com- Until recently it was generally believed that plex composed ofmRNA, an 80S ribosome and eukaryotic ribosomes initiate exclusively at met-tRNAi (l 3). Eukaryotic met-tRNAi differs AUG codons although it had been shown by from its prokaryotic counterpart by being asso- RAJBHANDARYand GHOSH (24) that yeast met- ciated with the 40S pre-initation complex before tRNAi may function with either AUG or GUG binding to mRNA (13).
    [Show full text]
  • Bio 102 Practice Problems Genetic Code and Mutation
    Bio 102 Practice Problems Genetic Code and Mutation Multiple choice: Unless otherwise directed, circle the one best answer: 1. Choose the one best answer: Beadle and Tatum mutagenized Neurospora to find strains that required arginine to live. Based on the classification of their mutants, they concluded that: A. one gene corresponds to one protein. B. DNA is the genetic material. C. "inborn errors of metabolism" were responsible for many diseases. D. DNA replication is semi-conservative. E. protein cannot be the genetic material. 2. Choose the one best answer. Which one of the following is NOT part of the definition of a gene? A. A physical unit of heredity B. Encodes a protein C. Segement of a chromosome D. Responsible for an inherited characteristic E. May be linked to other genes 3. A mutation converts an AGA codon to a TGA codon (in DNA). This mutation is a: A. Termination mutation B. Missense mutation C. Frameshift mutation D. Nonsense mutation E. Non-coding mutation 4. Beadle and Tatum performed a series of complex experiments that led to the idea that one gene encodes one enzyme. Which one of the following statements does not describe their experiments? A. They deduced the metabolic pathway for the synthesis of an amino acid. B. Many different auxotrophic mutants of Neurospora were isolated. C. Cells unable to make arginine cannot survive on minimal media. D. Some mutant cells could survive on minimal media if they were provided with citrulline or ornithine. E. Homogentisic acid accumulates and is excreted in the urine of diseased individuals. 5.
    [Show full text]
  • Eef2k) Natural Product and Synthetic Small Molecule Inhibitors for Cancer Chemotherapy
    International Journal of Molecular Sciences Review Progress in the Development of Eukaryotic Elongation Factor 2 Kinase (eEF2K) Natural Product and Synthetic Small Molecule Inhibitors for Cancer Chemotherapy Bin Zhang 1 , Jiamei Zou 1, Qiting Zhang 2, Ze Wang 1, Ning Wang 2,* , Shan He 1 , Yufen Zhao 2 and C. Benjamin Naman 1,* 1 Li Dak Sum Yip Yio Chin Kenneth Li Marine Biopharmaceutical Research Center, College of Food and Pharmaceutical Sciences, Ningbo University, Ningbo 315800, China; [email protected] (B.Z.); [email protected] (J.Z.); [email protected] (Z.W.); [email protected] (S.H.) 2 Institute of Drug Discovery Technology, Ningbo University, Ningbo 315211, China; [email protected] (Q.Z.); [email protected] (Y.Z.) * Correspondence: [email protected] (N.W.); [email protected] (C.B.N.) Abstract: Eukaryotic elongation factor 2 kinase (eEF2K or Ca2+/calmodulin-dependent protein kinase, CAMKIII) is a new member of an atypical α-kinase family different from conventional protein kinases that is now considered as a potential target for the treatment of cancer. This protein regulates the phosphorylation of eukaryotic elongation factor 2 (eEF2) to restrain activity and inhibit the elongation stage of protein synthesis. Mounting evidence shows that eEF2K regulates the cell cycle, autophagy, apoptosis, angiogenesis, invasion, and metastasis in several types of cancers. The Citation: Zhang, B.; Zou, J.; Zhang, expression of eEF2K promotes survival of cancer cells, and the level of this protein is increased in Q.; Wang, Z.; Wang, N.; He, S.; Zhao, many cancer cells to adapt them to the microenvironment conditions including hypoxia, nutrient Y.; Naman, C.B.
    [Show full text]
  • 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.
    [Show full text]
  • 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.
    [Show full text]
  • Role of Cyclin-Dependent Kinase 1 in Translational Regulation in the M-Phase
    cells Review Role of Cyclin-Dependent Kinase 1 in Translational Regulation in the M-Phase Jaroslav Kalous *, Denisa Jansová and Andrej Šušor Institute of Animal Physiology and Genetics, Academy of Sciences of the Czech Republic, Rumburska 89, 27721 Libechov, Czech Republic; [email protected] (D.J.); [email protected] (A.Š.) * Correspondence: [email protected] Received: 28 April 2020; Accepted: 24 June 2020; Published: 27 June 2020 Abstract: Cyclin dependent kinase 1 (CDK1) has been primarily identified as a key cell cycle regulator in both mitosis and meiosis. Recently, an extramitotic function of CDK1 emerged when evidence was found that CDK1 is involved in many cellular events that are essential for cell proliferation and survival. In this review we summarize the involvement of CDK1 in the initiation and elongation steps of protein synthesis in the cell. During its activation, CDK1 influences the initiation of protein synthesis, promotes the activity of specific translational initiation factors and affects the functioning of a subset of elongation factors. Our review provides insights into gene expression regulation during the transcriptionally silent M-phase and describes quantitative and qualitative translational changes based on the extramitotic role of the cell cycle master regulator CDK1 to optimize temporal synthesis of proteins to sustain the division-related processes: mitosis and cytokinesis. Keywords: CDK1; 4E-BP1; mTOR; mRNA; translation; M-phase 1. Introduction 1.1. Cyclin Dependent Kinase 1 (CDK1) Is a Subunit of the M Phase-Promoting Factor (MPF) CDK1, a serine/threonine kinase, is a catalytic subunit of the M phase-promoting factor (MPF) complex which is essential for cell cycle control during the G1-S and G2-M phase transitions of eukaryotic cells.
    [Show full text]