Auxiliary Factors Regulating Ribosome Function: Roles in Biogenesis, Antibiotic Resistance and Ribosome Recycling

Total Page:16

File Type:pdf, Size:1020Kb

Auxiliary Factors Regulating Ribosome Function: Roles in Biogenesis, Antibiotic Resistance and Ribosome Recycling Dissertation zur Erlangung des Doktorgrades der Fakultät für Chemie und Pharmazie der Ludwig-Maximilians-Universität München Auxiliary factors regulating ribosome function: Roles in biogenesis, antibiotic resistance and ribosome recycling Alexandra Dönhöfer aus München 2012 1 Erklärung Diese Dissertation wurde im Sinne von § 7 der Promotionsordnung vom 28. November 2011 von Herrn Prof. Dr. Roland Beckmann betreut. Eidesstattliche Versicherung Diese Dissertation wurde eigenständig und ohne unerlaubte Hilfe erarbeitet. München,................................ ....................................................... Alexandra Dönhöfer Dissertation eingereicht am: 09.02.2012 1. Gutachter: Herr Prof. Dr. Roland Beckmann 2. Gutachter: Herr Dr. Daniel N. Wilson Mündliche Prüfung am: 29.03.2012 2 Table of Contents LIST OF PUBLICATIONS ........................................................................................................7 CONTRIBUTION REPORT ......................................................................................................8 ABBREVIATIONS ..................................................................................................................10 1 INTRODUCTION .................................................................................................................13 1.1 The Flow of Genetic Information ................................................................................... 13 1.2 The Ribosome ................................................................................................................. 13 1.3 Ribosome Biogenesis in Eubacteria .............................................................................14 1.3.1 Processing of ribosomal RNA ....................................................................................15 1.3.2 Modification of ribosomal RNA ...................................................................................16 1.3.3 Modification of ribosomal Proteins ............................................................................. 19 1.3.4 Non-ribosomal Factors involved in Ribosome Assembly ............................................ 20 1.4 Ribosomal Proteins ........................................................................................................ 23 1.4.1 Ribosomal Proteins in Eubacteria ..............................................................................23 1.4.2 Ribosomal Proteins in Eukaryotes and Archaea ........................................................24 1.4.3 Specific Ribosomal Proteins in Organelles .................................................................25 1.4.3.1 Chloroplasts .........................................................................................................25 1.4.3.2 Mitochondria ........................................................................................................26 1.5 Translation Mechanism in Eubacteria...........................................................................27 1.5.1 Initiation Phase ........................................................................................................... 27 1.5.2 Elongation Phase: Decoding, Peptide-Bond Formation and Translocation ................ 29 1.5.3 Termination & Recycling ............................................................................................31 1.6 Inhibition of Translation: Antibiotics ............................................................................33 1.6.1 Stages of Inhibition during Translation .......................................................................35 1.6.2 Mechanisms of Antibiotic Resistance and Strategies to overcome this Problem ....... 37 3 2 OBJECTIVES AND AIMS OF THE THESIS .......................................................................40 3 CUMULATIVE THESIS: SUMMARY OF PUBLISHED RESULTS ..................................... 42 3.1 Paper I .............................................................................................................................. 42 3.2 Paper II ............................................................................................................................. 43 3.3 Paper III ............................................................................................................................ 44 3.4 Paper IV ........................................................................................................................... 45 3.5 Paper V ............................................................................................................................ 46 4 EXTRA-RIBOSOMAL FACTORS IN RIBOSOME BIOGENESIS ....................................... 48 4.1 Project Aims .................................................................................................................... 48 4.2 Introduction ..................................................................................................................... 48 4.2.1 Post-transcriptional Modification by Methyltransferases............................................. 49 4.2.2 Guanine 966 specific Methyltransferase YhhF (RsmD)..............................................50 4.2.3 Ψ1915 specific Methyltransferase RlmH (YbeA) ........................................................51 4.2.4 Ribosome maturation factor M (RimM) .......................................................................52 4.3 Results ............................................................................................................................. 54 4.3.1 Purification of E. coli YhhF protein .............................................................................54 4.3.2 In vitro binding assay: Eco30SyhhF + YhhF + sinefungin ........................................ 55 4.3.3 3D-reconstruction of the [Eco30SyhhF·YhhF] complex ........................................... 56 4.3.4 In vitro binding assay: Eco70SrluD/rlmH + RlmH + sinefungin ................................ 57 4.3.5 3D-reconstruction of the [Eco70SrluD/rlmH·RlmH] complex .................................... 59 4.3.6 Ribosome profiles and purification of E. coli RimM .................................................... 61 4.3.7 In vitro binding assay: Eco30S/Eco30SrimM + RimM .............................................. 62 4.3.8 3D-reconstruction of the [Eco30SrimM·RimM] complex ........................................... 63 4.4 Conclusions and Outlook ..............................................................................................65 5 ORGANELLAR-SPECIFIC RIBOSOMAL PROTEINS ........................................................67 4 5.1 Project Aims .................................................................................................................... 67 5.2 Introduction ..................................................................................................................... 67 5.2.1 Mitochondrion-specific ribosomal proteins (MSRPs) ..................................................67 5.3 Results ............................................................................................................................. 68 5.3.1 Expression and purification of yeast MSRPs ..............................................................68 5.3.2 In vivo association of individual MSRPs with E. coli ribosomes ................................. 70 5.3.3 Purification of [Eco70S·MRPL3] complexes ...............................................................72 5.3.4 3D-reconstruction of [Eco70S·MRPL3] complexes ....................................................73 5.4 Conclusions and Outlook ..............................................................................................74 6 ANTIBIOTIC RESISTANCE PROTEINS .............................................................................76 6.1 Project Aims .................................................................................................................... 76 6.2 Introduction ..................................................................................................................... 76 6.2.1 Ribosome protection protein Tet(M) ...........................................................................76 6.3 Results ............................................................................................................................. 78 6.3.1 Purification of E. faecalis Tet(M) ................................................................................78 6.3.2 In vitro binding assay: Eco70S + Tet(M) + GDPNP +/- Tgc ....................................... 79 6.3.3 3D-reconstruction of [Eco70S·Tet(M)·GDPNP·Tgc] complex .................................... 81 6.4 Conclusions and Outlook ............................................................................................101 7 MATERIAL AND METHODS .............................................................................................106 7.1 Methods in Molecular Biology .....................................................................................106 7.1.1 Media and Supplements ...........................................................................................106 7.1.2 Strains ...................................................................................................................... 106 7.1.3 Polymerase Chain Reaction (PCR) ..........................................................................106 7.1.4 Agarose Gel Electrophoresis ....................................................................................108 7.1.5 Cloning
Recommended publications
  • Analysis of Gene Expression Data for Gene Ontology
    ANALYSIS OF GENE EXPRESSION DATA FOR GENE ONTOLOGY BASED PROTEIN FUNCTION PREDICTION A Thesis Presented to The Graduate Faculty of The University of Akron In Partial Fulfillment of the Requirements for the Degree Master of Science Robert Daniel Macholan May 2011 ANALYSIS OF GENE EXPRESSION DATA FOR GENE ONTOLOGY BASED PROTEIN FUNCTION PREDICTION Robert Daniel Macholan Thesis Approved: Accepted: _______________________________ _______________________________ Advisor Department Chair Dr. Zhong-Hui Duan Dr. Chien-Chung Chan _______________________________ _______________________________ Committee Member Dean of the College Dr. Chien-Chung Chan Dr. Chand K. Midha _______________________________ _______________________________ Committee Member Dean of the Graduate School Dr. Yingcai Xiao Dr. George R. Newkome _______________________________ Date ii ABSTRACT A tremendous increase in genomic data has encouraged biologists to turn to bioinformatics in order to assist in its interpretation and processing. One of the present challenges that need to be overcome in order to understand this data more completely is the development of a reliable method to accurately predict the function of a protein from its genomic information. This study focuses on developing an effective algorithm for protein function prediction. The algorithm is based on proteins that have similar expression patterns. The similarity of the expression data is determined using a novel measure, the slope matrix. The slope matrix introduces a normalized method for the comparison of expression levels throughout a proteome. The algorithm is tested using real microarray gene expression data. Their functions are characterized using gene ontology annotations. The results of the case study indicate the protein function prediction algorithm developed is comparable to the prediction algorithms that are based on the annotations of homologous proteins.
    [Show full text]
  • Rps3/Us3 Promotes Mrna Binding at the 40S Ribosome Entry Channel
    Rps3/uS3 promotes mRNA binding at the 40S ribosome PNAS PLUS entry channel and stabilizes preinitiation complexes at start codons Jinsheng Donga, Colin Echeverría Aitkenb, Anil Thakura, Byung-Sik Shina, Jon R. Lorschb,1, and Alan G. Hinnebuscha,1 aLaboratory of Gene Regulation and Development, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892; and bLaboratory on the Mechanism and Regulation of Protein Synthesis, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892 Contributed by Alan G. Hinnebusch, January 24, 2017 (sent for review December 15, 2016; reviewed by Jamie H. D. Cate and Matthew S. Sachs) Met The eukaryotic 43S preinitiation complex (PIC) bearing Met-tRNAi rearrangement to PIN at both near-cognate start codons (e.g., in a ternary complex (TC) with eukaryotic initiation factor (eIF)2-GTP UUG) and cognate (AUG) codons in poor Kozak context; hence scans the mRNA leader for an AUG codon in favorable “Kozak” eIF1 must dissociate from the 40S subunit for start-codon rec- context. AUG recognition provokes rearrangement from an open ognition (Fig. 1A). Consistent with this, structural analyses of PIC conformation with TC bound in a state not fully engaged with partial PICs reveal that eIF1 and eIF1A promote rotation of the “ ” the P site ( POUT ) to a closed, arrested conformation with TC tightly 40S head relative to the body (2, 3), thought to be instrumental bound in the “P ” state. Yeast ribosomal protein Rps3/uS3 resides IN in TC binding in the POUT conformation, but that eIF1 physically in the mRNA entry channel of the 40S subunit and contacts mRNA Met clashes with Met-tRNAi in the PIN state (2, 4), and is both via conserved residues whose functional importance was unknown.
    [Show full text]
  • The Analysis of Translation-Related Gene Set
    The analysis of translation-related gene set boosts debates around origin and evolution of mimiviruses Jonatas Santos Abrahao, Rodrigo Araujo, Philippe Colson, Bernard La Scola To cite this version: Jonatas Santos Abrahao, Rodrigo Araujo, Philippe Colson, Bernard La Scola. The analysis of translation-related gene set boosts debates around origin and evolution of mimiviruses. PLoS Ge- netics, Public Library of Science, 2017, 13 (2), 10.1371/journal.pgen.1006532. hal-01496184 HAL Id: hal-01496184 https://hal.archives-ouvertes.fr/hal-01496184 Submitted on 7 May 2018 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. REVIEW The analysis of translation-related gene set boosts debates around origin and evolution of mimiviruses JoÃnatas Santos Abrahão1,2☯, Rodrigo Arau jo2☯, Philippe Colson1, Bernard La Scola1* 1 Unite de Recherche sur les Maladies Infectieuses et Tropicales Emergentes (URMITE) UM63 CNRS 7278 IRD 198 INSERM U1095, Aix-Marseille Univ., 27 boulevard Jean Moulin, Faculte de MeÂdecine, Marseille, France, 2 Instituto de Ciências BioloÂgicas, Departamento de Microbiologia, LaboratoÂrio de VõÂrus, Universidade Federal de Minas Gerais, Belo Horizonte, Brazil ☯ These authors contributed equally to this work. * [email protected] Abstract a1111111111 a1111111111 The giant mimiviruses challenged the well-established concept of viruses, blurring the roots a1111111111 of the tree of life, mainly due to their genetic content.
    [Show full text]
  • The Microbiota-Produced N-Formyl Peptide Fmlf Promotes Obesity-Induced Glucose
    Page 1 of 230 Diabetes Title: The microbiota-produced N-formyl peptide fMLF promotes obesity-induced glucose intolerance Joshua Wollam1, Matthew Riopel1, Yong-Jiang Xu1,2, Andrew M. F. Johnson1, Jachelle M. Ofrecio1, Wei Ying1, Dalila El Ouarrat1, Luisa S. Chan3, Andrew W. Han3, Nadir A. Mahmood3, Caitlin N. Ryan3, Yun Sok Lee1, Jeramie D. Watrous1,2, Mahendra D. Chordia4, Dongfeng Pan4, Mohit Jain1,2, Jerrold M. Olefsky1 * Affiliations: 1 Division of Endocrinology & Metabolism, Department of Medicine, University of California, San Diego, La Jolla, California, USA. 2 Department of Pharmacology, University of California, San Diego, La Jolla, California, USA. 3 Second Genome, Inc., South San Francisco, California, USA. 4 Department of Radiology and Medical Imaging, University of Virginia, Charlottesville, VA, USA. * Correspondence to: 858-534-2230, [email protected] Word Count: 4749 Figures: 6 Supplemental Figures: 11 Supplemental Tables: 5 1 Diabetes Publish Ahead of Print, published online April 22, 2019 Diabetes Page 2 of 230 ABSTRACT The composition of the gastrointestinal (GI) microbiota and associated metabolites changes dramatically with diet and the development of obesity. Although many correlations have been described, specific mechanistic links between these changes and glucose homeostasis remain to be defined. Here we show that blood and intestinal levels of the microbiota-produced N-formyl peptide, formyl-methionyl-leucyl-phenylalanine (fMLF), are elevated in high fat diet (HFD)- induced obese mice. Genetic or pharmacological inhibition of the N-formyl peptide receptor Fpr1 leads to increased insulin levels and improved glucose tolerance, dependent upon glucagon- like peptide-1 (GLP-1). Obese Fpr1-knockout (Fpr1-KO) mice also display an altered microbiome, exemplifying the dynamic relationship between host metabolism and microbiota.
    [Show full text]
  • Translation Termination and Ribosome Recycling in Eukaryotes
    Downloaded from http://cshperspectives.cshlp.org/ on October 3, 2021 - Published by Cold Spring Harbor Laboratory Press Translation Termination and Ribosome Recycling in Eukaryotes Christopher U.T. Hellen Department of Cell Biology, State University of New York, Downstate Medical Center, New York, New York 11203 Correspondence: [email protected] Termination of mRNA translation occurs when a stop codon enters the A site of the ribosome, and in eukaryotes is mediated by release factors eRF1 and eRF3, which form a ternary eRF1/ eRF3–guanosine triphosphate (GTP) complex. eRF1 recognizes the stop codon, and after hydrolysis of GTP by eRF3, mediates release of the nascent peptide. The post-termination complex is then disassembled, enabling its constituents to participate in further rounds of translation. Ribosome recycling involves splitting of the 80S ribosome by the ATP-binding cassette protein ABCE1 to release the 60S subunit. Subsequent dissociation of deacylated transfer RNA (tRNA) and messenger RNA (mRNA) from the 40S subunit may be mediated by initiation factors (priming the 40S subunit for initiation), by ligatin (eIF2D) or by density- regulated protein (DENR) and multiple copies in T-cell lymphoma-1 (MCT1). These events may be subverted by suppression of termination (yielding carboxy-terminally extended read- through polypeptides) or by interruption of recycling, leading to reinitiation of translation near the stop codon. OVERVIEW OF TRANSLATION post-termination complex (post-TC) is recycled TERMINATION AND RECYCLING bysplittingoftheribosome,whichismediatedby ABCE1. This step is followed by release of de- ranslation is a cyclical process that comprises acylated tRNA and messenger RNA (mRNA) Tinitiation, elongation, termination, and ribo- from the 40S subunit via redundant pathways some recycling stages (Jackson et al.
    [Show full text]
  • Eif1 Discriminates Against Suboptimal Initiation Sites to Prevent Excessive Uorf Translation Genome-Wide
    Downloaded from rnajournal.cshlp.org on September 24, 2021 - Published by Cold Spring Harbor Laboratory Press eIF1 discriminates against suboptimal initiation sites to prevent excessive uORF translation genome-wide FUJUN ZHOU, HONGEN ZHANG, SHARDUL D. KULKARNI, JON R. LORSCH, and ALAN G. HINNEBUSCH Division of Molecular and Cellular Biology, Eunice Kennedy Shriver National Institute of Child Health and Development, National Institutes of Health, Bethesda, Maryland 20892, USA ABSTRACT The translation preinitiation complex (PIC) scans the mRNA for an AUG codon in a favorable context. Previous findings sug- gest that the factor eIF1 discriminates against non-AUG start codons by impeding full accommodation of Met-tRNAi in the P site of the 40S ribosomal subunit, necessitating eIF1 dissociation for start codon selection. Consistent with this, yeast eIF1 substitutions that weaken its binding to the PIC increase initiation at UUG codons on a mutant his4 mRNA and par- ticular synthetic mRNA reporters; and also at the AUG start codon of the mRNA for eIF1 itself owing to its poor Kozak con- text. It was not known however whether such eIF1 mutants increase initiation at suboptimal start codons genome-wide. By ribosome profiling, we show that the eIF1-L96P variant confers increased translation of numerous upstream open reading frames (uORFs) initiating with either near-cognate codons (NCCs) or AUGs in poor context. The increased uORF translation is frequently associated with the reduced translation of the downstream main coding sequences (CDS). Initiation is also elevated at certain NCCs initiating amino-terminal extensions, including those that direct mitochondrial localization of the GRS1 and ALA1 products, and at a small set of main CDS AUG codons with especially poor context, including that of eIF1 itself.
    [Show full text]
  • Translation Initiation: Structures, Mechanisms and Evolution
    Quarterly Reviews of Biophysics 37, 3/4 (2004), pp. 197–284. f 2004 Cambridge University Press 197 doi:10.1017/S0033583505004026 Printed in the United Kingdom Translationinitiation: structures, mechanisms and evolution Assen Marintchev and Gerhard Wagner* Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, USA Abstract. Translation, the process of mRNA-encoded protein synthesis, requires a complex apparatus, composed of the ribosome, tRNAs and additional protein factors, including aminoacyl tRNA synthetases. The ribosome provides the platform for proper assembly of mRNA, tRNAs and protein factors and carries the peptidyl-transferase activity. It consists of small and large subunits. The ribosomes are ribonucleoprotein particles with a ribosomal RNA core, to which multiple ribosomal proteins are bound. The sequence and structure of ribosomal RNAs, tRNAs, some of the ribosomal proteins and some of the additional protein factors are conserved in all kingdoms, underlying the common origin of the translation apparatus. Translation can be subdivided into several steps: initiation, elongation, termination and recycling. Of these, initiation is the most complex and the most divergent among the different kingdoms of life. A great amount of new structural, biochemical and genetic information on translation initiation has been accumulated in recent years, which led to the realization that initiation also shows a great degree of conservation throughout evolution. In this review, we summarize the available structural and functional data on translation initiation in the context of evolution, drawing parallels between eubacteria, archaea, and eukaryotes. We will start with an overview of the ribosome structure and of translation in general, placing emphasis on factors and processes with relevance to initiation.
    [Show full text]
  • Competition Between Translation Initiation Factor Eif5 and Its Mimic
    Published online 18 September 2017 Nucleic Acids Research, 2017, Vol. 45, No. 20 11941–11953 doi: 10.1093/nar/gkx808 Competition between translation initiation factor eIF5 and its mimic protein 5MP determines non-AUG initiation rate genome-wide Leiming Tang1,†, Jacob Morris1,†,JiWan2,†, Chelsea Moore1,†, Yoshihiko Fujita3,†, Sarah Gillaspie1,†, Eric Aube1, Jagpreet Nanda4, Maud Marques5, Maika Jangal5, Abbey Anderson1, Christian Cox1, Hiroyuki Hiraishi1, Leiming Dong2, Hirohide Saito3, Chingakham Ranjit Singh1, Michael Witcher5, Ivan Topisirovic5, Shu-Bing Qian2 and Katsura Asano1,* 1Molecular Cellular and Developmental Biology Program, Division of Biology, Kansas State University, Manhattan, KS 66506, USA, 2Division of Nutritional Sciences, Cornell University, Ithaca, NY 14853, USA, 3Center for iPS Cell Research and Application, Kyoto University, Sakyo-ku, Kyoto 606-8507, Japan, 4NIGMS, NIH, Bethesda, MD 20892, USA and 5Lady Davis Institute, and the Gerald Bronfman Department of Oncology, McGill University, Montreal, QC H3A 2B4, Canada Received July 16, 2017; Revised August 25, 2017; Editorial Decision August 28, 2017; Accepted August 31, 2017 ABSTRACT For eukaryotic translation initiation to proceed, the cap- binding complex eIF4F must bind to m7G-capped mRNAs In the human genome, translation initiation from non- and recruit them to the 40S small ribosomal subunit (SSU) AUG codons plays an important role in various gene (1). Prior to this event, the 40S SSU is activated into an regulation programs. However, mechanisms regulat- open, scanning-competent form through eukaryotic initia- Met ing the non-AUG initiation rate remain poorly under- tion factors bound to Met-tRNAi , allowing formation stood. Here, we show that the non-AUG initiation rate of the 43S ribosome pre-initiation complex (PIC) (2).
    [Show full text]
  • Universally Conserved Translation Initiation Factors
    Proc. Natl. Acad. Sci. USA Vol. 95, pp. 224–228, January 1998 Evolution Universally conserved translation initiation factors NIKOS C. KYRPIDES* AND CARL R. WOESE Department of Microbiology, University of Illinois at Urbana-Champaign, B103 Chemistry and Life Sciences, MC 110, 407 South Goodwin, Urbana, IL 61801 Contributed by Carl R. Woese, November 14, 1997 ABSTRACT The process by which translation is initiated mRNAs are polycistronic, uncapped, lack long poly(A) tails, has long been considered similar in Bacteria and Eukarya but and have Shine–Dalgarno sequences suggested (erroneously) accomplished by a different unrelated set of factors in the two that the archaeal process resembled the bacterial one. Yet, the cases. This not only implies separate evolutionary histories for M. jannaschii genome showed that archaeal translation initi- the two but also implies that at the universal ancestor stage, ation is remarkably similar to that seen in eukaryotes (10). a translation initiation mechanism either did not exist or was Homologs of eukaryotic factors eIF-1A, eIF-2 (all three of a different nature than the extant processes. We demon- subunits), two of the five eIF-2B subunits (a and d), eIF-4A, strate herein that (i) the ‘‘analogous’’ translation initiation and eIF-5A were reported (10), a list that covers most eu- factors IF-1 and eIF-1A are actually related in sequence, (ii) karyotic factors (except for those involved with mRNA cap the ‘‘eukaryotic’’ translation factor SUI1 is universal in recognition). distribution, and (iii) the eukaryoticyarchaeal translation The three recognized bacterial translation initiation factors factor eIF-5A is homologous to the bacterial translation factor have functional counterparts among the eukaryotic factors— EF-P.
    [Show full text]
  • “Ribozooming” – Initiating, Terminating and Controlling Protein Synthesis
    Teze disertace k získání vědeckého titulu „doktor věd“ ve skupině molekulárně-biologických a lékařských věd. “Ribozooming” – initiating, terminating and controlling protein synthesis. Komise pro obhajoby doktorských disertací v oboru: molekulární biologie a genetika Leoš Shivaya Valášek Mikrobiologický ústav AVČR, v.v.i. Jeřmanice, 30. dubna 2016 1 TABLE OF CONTENT Summary…………………………………………………………………………….……..…3 Souhrn……………………………………………………………………………….………..4 Introduction………………………………………………………………………….……..…5 Translation initiation and control in eukaryotes……………..………………….……..….6 Translation termination and stop codon readthrough in eukaryotes………………….10 Author’s contribution to the field set in the historical perspective…………….…….....12 Conclusions...………………………………………………………………………..…..….35 References………………………………………………………………………….……....36 List of the DSc thesis publications……………………………………………….….……45 List of other publications by the author…………………………………………………..49 2 SUMMARY Protein synthesis is a fundamental biological mechanism bringing the DNA-encoded genetic information into life by its translation into molecular effectors - proteins. The initiation phase of translation is one of the key points of regulation of gene expression in eukaryotes, playing a role in numerous processes from development to aging. Translation termination is also a subject of translational control via so called programmed stop codon readthrough that increases a variability of the proteome by extending C-termini of the selected proteins, for example upon stress. Indeed, the importance
    [Show full text]
  • Eif1a Residues Implicated in Cancer Stabilize Translation Preinitiation 6 Complexes and Favor Suboptimal Initiation Sites in Yeast
    1 2 3 4 5 eIF1A residues implicated in cancer stabilize translation preinitiation 6 complexes and favor suboptimal initiation sites in yeast 7 1,2 3 3 1 1 3 8 Pilar Martin-Marcos , Fujun Zhou , Charm Karunasiri , Fan Zhang , Jinsheng Dong , Jagpreet Nanda , 9 Neelam Sen1, Mercedes Tamame2, Michael Zeschnigk4, Jon R. Lorsch3† and Alan G. Hinnebusch1† 10 11 12 1Laboratory of Gene Regulation and Development, Eunice Kennedy Shriver National Institute of Child 13 Health and Development, National Institutes of Health, Bethesda, MD 20892 14 2Instituto de Biología Funcional y Genómica, IBFG-CSIC. Universidad de Salamanca, Salamanca, Spain 15 3Laboratory on the Mechanism and Regulation of Protein Synthesis, Eunice Kennedy Shriver National 16 Institute of Child Health and Development, National Institutes of Health, Bethesda, MD 20892 17 4Institute of Human Genetics & Eye Cancer Research Group, Faculty of Medicine, University Duisburg- 18 Essen, Essen, Germany. 19 20 †Corresponding authors: [email protected]; [email protected] 21 Keywords: translation, initiation, eIF1A, uveal melanoma, yeast 22 Running title: UM-associated eIF1A mutations increase initiation accuracy 23 1 24 25 ABSTRACT 26 The translation pre-initiation complex (PIC) scans the mRNA for an AUG codon in favorable 27 context, and AUG recognition stabilizes a closed PIC conformation. The unstructured N-terminal 28 tail (NTT) of yeast eIF1A deploys five basic residues to contact tRNAi, mRNA, or 18S rRNA exclusively 29 in the closed state. Interestingly, EIF1AX mutations altering the human eIF1A NTT are associated with 30 uveal melanoma (UM). We found that substituting all five basic residues, and seven UM-associated 31 substitutions, in yeast eIF1A suppresses initiation at near-cognate UUG codons and AUGs in poor 32 context.
    [Show full text]
  • Initiation Context Modulates Autoregulation of Eukaryotic Translation Initiation Factor 1 (Eif1)
    Initiation context modulates autoregulation of eukaryotic translation initiation factor 1 (eIF1) Ivaylo P. Ivanova,b,1,2, Gary Loughrana,1, Matthew S. Sachsc, and John F. Atkinsa,b,2 aBioSciences Institute, University College Cork, Cork, Ireland; bDepartment of Human Genetics, University of Utah, Salt Lake City, UT 84112-5330; and cDepartment of Biology, Texas A&M University, College Station, TX 77843 Edited by Jonathan S. Weissman, University of California, San Francisco, CA, and approved September 20, 2010 (received for review June 30, 2010) The central feature of standard eukaryotic translation initiation is Autoregulation of the synthesis of translation factors is often small ribosome subunit loading at the 5′ cap followed by its 5′ to 3′ observed (17). Initiation of IF3 translation begins with an AUU scanning for a start codon. The preferred start is an AUG codon in an codon that is an important component of a negative feedback optimal context. Elaborate cellular machinery exists to ensure the regulatory loop. High levels of IF3 protein increase discrimination fidelity of start codon selection. Eukaryotic initiation factor 1 (eIF1) against initiation at its AUU start codon, which leads to reduced plays a central role in this process. Here we show that the translation synthesis of the protein (18, 19). The present work addresses of eIF1 homologs in eukaryotes from diverse taxa involves initiation whether a counterpart autoregulatory mechanism controls initia- from an AUG codon in a poor context. Using human eIF1 as a model, tion at the eIF1 start codon. we show that this poor context is necessary for an autoregulatory negative feedback loop in which a high level of eIF1 inhibits its own Results translation, establishing that variability in the stringency of start co- The Initiation Codon of eIF1 in Diverse Eukaryotes Has Poor Initiation don selection is used for gene regulation in eukaryotes.
    [Show full text]