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Translation Mechanism in Production of Cocksfoot Mottle Virus Proteins Recent Publications in this Series: MÄKELÄINEN KATRI MÄKELÄINEN KATRI View metadata, citation and similar papers at core.ac.uk brought to you by CORE 6/2005 Sanna Edelman provided by Helsingin yliopiston digitaalinen arkisto Mucosa-Adherent Lactobacilli: Commensal and Pathogenic Characteristics 7/2005 Leena Karhinen Glycosylation and Sorting Of Secretory Proteins in the Endoplasmic Reticulum of the Yeast Saccharomyces cerevisiae 8/2005 Saurabh Sen Functional Studies on alpha2-Adrenergic Receptor Subtypes Proteins Virus of Cocksfoot Mottle Mechanisms in Production Translation Translation: Lost in Proteins Virus of Cocksfoot Mottle Mechanisms in Production Translation Translation: Lost in 9/2005 Tiina E. Raevaara Functional Significance of Minor MLH1 Germline Alterations Found in Colon Cancer Patients 10/2005 Katja Pihlainen Liquid Chromatography and Atmospheric Pressure Ionisation Mass Spectrometry in Analysing Drug Seizures 11/2005 Pietri Puustinen Posttranslational Modifications of Potato Virus A Movement Related Proteins CP and VPg 12/2005 Irmgard Suominen Paenibacillus and Bacillus Related to Paper and Spruce Tree 13/2005 Heidi Hyytiäinen Regulatory Networks Controlling Virulence in the Plant Pathogen Erwinia Carotovora Ssp. Carotovora 14/2005 Sanna Janhunen Lost in Translation: Translation Mechanisms in Different Responses of the Nigrostriatal and Mesolimbic Dopaminergic Pathways to Nicotinic Receptor Agonists 15/2005 Denis Kainov Packaging Motors of Cystoviruses Production of Cocksfoot Mottle Virus Proteins 16/2005 Ivan Pavlov Heparin-Binding Growth-Associated Molecule (HB-GAM) in Activity-Dependent Neuronal Plasticity in Hippocampus 17/2005 Laura Seppä Regulation of Heat Shock Response in Yeast and Mammalian Cells 18/2005 Veli-Pekka Jaakola Functional and Structural Studies on Heptahelical Membrane Proteins 19/2005 Anssi Rantakari Characterisation of the Type Three Secretion System in Erwinia carotovora 20/2005 Sari Airaksinen Role of Excipients in Moisture Sorption and Physical Stability of Solid Pharmaceutical Formulations KATRI MÄKELÄINEN 21/2005 Tiina Hilden Affinity and Avidity of the LFA-1 Integrin is Regulated by Phosphorylation 22/2005 Ari Pekka Mähönen Cytokinins Regulate Vascular Morphogenesis in the Arabidopsis thaliana Root 23/2005 J. Matias Palva Institute of Biotechnology and Interactions Among Neuronal Oscillations in the Developing and Adult Brain Department of Applied Biology 25/2005 Michael Stefanidakis Cell-Surface Association between Progelatinases and ß2 Integrins: Role of the Complexes in Leukocyte Department of Applied Chemistry and Microbiology Migration 26/2005 Heli Kansanaho Faculty of Agriculture and Forestry, and Implementation of the Principles of Patient Counselling into Practice in Finnish Community Pharmacies Viikki Graduate School in Biosciences 1/2006 Julia Perttilä Expression, Enzymatic Activities and Subcellular Localization of Hepatitis E Virus and Semliki Forest Virus University of Helsinki Replicase Proteins 2/2006 Tero Wennberg Computer-Assisted Separation and Primary Screening of Bioactive Compounds Helsinki 2006 ISSN 1795-7079 ISBN 952-10-3016-X Dissertationes bioscientiarum molecularium Universitatis Helsingiensis in Viikki 3/2006 3/2006 3/2006 Lost in Translation: Translation Mechanisms in Production of Cocksfoot Mottle Virus Proteins Katri Mäkeläinen Institute of Biotechnology and Department of Applied Biology Department of Applied Chemistry and Microbiology Faculty of Agriculture and Forestry, and Viikki Graduate School in Biosciences University of Helsinki ACADEMIC DISSERTATION To be presented, with the permission of the Faculty of Agriculture and Forestry of the University of Helsinki, for public criticism in the auditorium 1041 at Viikki Biocenter (Viikinkaari 5) on March 31th, 2006, at 12 noon. Supervisor: Docent Kristiina Mäkinen Department of Applied Chemistry and Microbiology University of Helsinki Finland Reviewers: Docent Maija Vihinen-Ranta Department of Biological and Environmental Science University of Jyväskylä Finland And Professor Carl-Henrik von Bonsdorff Department of Food and Environmental Hygiene University of Helsinki Finland Opponent: Professor W. Allen Miller Plant Pathology Department Iowa State University USA 3/2006 ISBN 952-10-3016-X (paperback) ISBN 952-10-3017-8 (PDF, online) ISSN 1795-7079 (paperback) ISSN 1795-8229 (PDF, online) Gummerus Kirjapaino Oy Vaajakoski 2006 To my family ORIGINAL PUBLICATIONS I Mäkeläinen K., Aspegren K., Wahlström E., Teeri T.H., and Mäkinen K. Comparison of the translational properties of Cocksfoot mottle virus 5’leader sequence with known translational enhancers from other plant viruses. (Manuscript) II Mäkeläinen K., and Mäkinen K. Testing of internal initiation via dicistronic constructs is complicated by production of extraneous transcripts. (Submitted) III Mäkinen K., Mäkeläinen K., Arshava N., Tamm T., Merits A., Truve E., Zavriev S., and Saarma M. 2000. Characterization of VPg and the polyprotein processing of Cocksfoot mottle virus (genus Sobemovirus). J. Gen. Virol. 81: 2783-2789. IV Lucchesi J., Mäkeläinen K., Merits A., Tamm T., and Mäkinen K. 2000. Regulation of –1 ribosomal frameshifting directed by Cocksfoot mottle sobemovirus genome. Eur. J. Biochem. 267: 3523-3529. V Mäkeläinen K., and Mäkinen K. 2005. Factors affecting translation at the programmed –1 ribosomal frameshifting site of Cocksfoot mottle virus RNA in vivo. Nucleic Acids Res. 33: 2239-2247. Also unpublished data will be presented. CONTENTS LIST OF ORIGINAL PUBLICATIONS ABBREVIATIONS ABSTRACT ................................................................................................................ 1 1. INTRODUCTION ................................................................................................... 2 1.1 TRANSLATION INITIATION ..................................................................... 3 1.1.1 Regulation of translation initiation during virus infection .......................6 1.1.2 Plant viral translational enhancers ........................................................... 7 1.1.3 Internal ribosome entry sites (IRESs) ..................................................... 9 1.1.3.1 Mechanism of IRES-mediated translation initiation ................. 10 1.1.3.2 IRES trans-acting factors (ITAFs) ............................................ 11 1.1.4 Leaky scanning ...................................................................................... 12 1.1.5 Reinitiation ............................................................................................ 12 1.1.6 SgRNAs ................................................................................................. 13 1.2 TRANSLATION ELONGATION ............................................................... 13 1.2.1 Regulation of elongation ....................................................................... 15 1.2.2 Programmed -1 ribosomal frameshifting (-1 PRF) ............................... 16 1.2.2.1 The -1 PRF signals .................................................................... 17 1.2.2.2 Mechanism of -1 PRF ............................................................... 19 1.3 TERMINATION OF TRANSLATION ...................................................... 21 1.3.1 Programmed termination codon readthrough ........................................ 22 1.4 POSTTRANSLATIONAL REGULATION OF GENE EXPRESSION .. 22 1.4.1 Polyprotein processing .......................................................................... 23 1.4.2 Viral proteases ....................................................................................... 24 2. AIMS OF THE STUDY ........................................................................................ 26 3. MATERIALS AND METHODS .......................................................................... 27 4. RESULTS AND DISCUSSION ............................................................................ 31 4.1 Translation initiation from CfMV RNA .................................................... 31 4.1.1 Comparison of protein production from CfMVε with known plant viral translational enhancers (I) ............................................................. 31 4.1.2 Identifi cation of regions important for gene expression from CfMVε in barley suspension cells (I) ............................................................. 32 4.1.3 Transient expression from in vitro transcribed mRNAs (I) ................... 34 4.1.4 Functioning of viral leader sequences in S. cerevisiae (II) ................... 34 4.1.5 Translational properties of CfMVε in vitro (I) ...................................... 35 4.1.6 Contribution of CfMV 3’UTR on translation initiation from CfMVε (I, unpublished) ................................................................................. 37 4.2 Does CfMVε promote internal initiation of translation? ......................... 38 4.2.1 Studies on internal initiation in WGE (I) .............................................. 38 4.2.2 Internal initiation in barley suspension cells (unpublished) .................. 42 4.2.3 Internal initiation in yeast (II) ............................................................... 43 4.2.3.1 Identifi cation of regions important for gene expression from internally positioned CfMVε in yeast (unpublished) ..............................48 4.2.3.2 Determination of 3’ cistron translation from dicistronic mRNAs in yeast spheroplasts (II) ......................................................... 49 4.2.4 Evaluation of the dicistronic approach in IRES
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