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Studies on Translation Initiation and Termination in Escherichia Coli Studies on translation initiation and termination in Escherichia coli Georgina Ibrahim Isak Department of Genetics, Microbiology and Toxicology Stockholm University, Sweden 2012 Doctoral thesis 2012 Department of Genetics, Microbiology and Toxicology Stockholm University SE-106 91, Sweden ©Georgina Ibrahim Isak, Stockholm 2012 ISBN 978-91-7447-388-9 Printed in Sweden by Universitetsservice US-AB, Stockholm 2012 Distributor: Department of Genetics, Microbiology and Toxicology Cover illustration: Structure of the ribosome. The 70S ribosome with mRNA and A- P- and E- site tRNAs. Picture adapted with permission from [1]. Copyright © 2009, Rights Managed by Nature Publishing Group. 2 Till min familj 3 4 Abstract Translation initiation factor 1 (IF1) has been shown to be an RNA chaperone. In order to find functional interactions that IF1 may have with rRNA, we have isolated second‐site suppressors of a cold‐sensitive IF1 mutant. Joining of the ribosomal subunit seems to be affected in the IF1 mutant strain and the suppressive effect is a consequence of decreasing the available pool of mature 50S subunits. The results serve as additional evidence that IF1 is an RNA chaperone and that final maturation of the ribosome takes place during translation initiation. In this study we have also investigated the effect of a cold‐sensitive mutant IF1 or kasugamycin addition on gene expression using a 2D gel electrophoresis technique. The effect is much more dramatic when cells are treated with kasugamycin compared to mutant IF1. The ybgF gene is uniquely sensitive to the IF1 mutation as well as the addition of kasugamycin. This effect on the native gene could be connected with some property of the TIR sequence of ybgF and supports the notion that kasugamycin addition and the IF1 cold‐sensitive mutation have a similar TIR‐specific effect on mRNA translation. Finally we have isolated a suppressor of a temperature‐sensitive mutation in ribosomal release factor 1 (RF1) to shed more light on the translation termination process. The suppressor mutation is linked to an IS10 insertion into the cysB gene and results in a Cys‐ phenotype. Our results suggest that suppression of the thermosensitive growth is a consequence of the mnm5s2U hypomodification of certain tRNA species. The ability of mnm5s2U hypomodified tRNA to induce frameshifting may be responsible for the suppression mechanism and it supports the hypothesis that modified nucleosides in the anticodon of tRNA act in part to prevent frameshifting by the ribosome. 5 List of publications The thesis is based on the following publications I. Jaroslav Belotserkovsky, Georgina Isak, Leif A. Isaksson (2011) Suppression of a cold‐sensitive mutant initiation factor 1 by alterations in the 23S rRNA maturation region. FEBS J., 278(10):1745‐56 II. Sergey Surkov, Georgina Isak, Leif A. Isaksson Influences of a mutated translation initiation factor IF1 or kasugamycin on Escherichia coli gene expression. (Submitted) III. Georgina Isak, Monica Rydén‐Aulin (2009) Hypomodification of the wobble base in tRNAGlu, tRNALys, and tRNAGln suppresses the temperature‐sensitive phenotype caused by mutant release factor 1. J. Bacteriol.,191(5):1604‐9 Permissions to reproduce papers I and III were kindly obtained from the publishers 6 Table of contents 1. Introduction…………………………………………………………………………... 9 1.1 Bacterial translation process……………………………………………………….. 9 1.2 The Bacterial ribosome and its subunits………………………………………….. 10 1.2.1 The small ribosomal subunit………………………………………………....... 11 1.2.2 The large ribosomal subunit…………………………………………………… 11 1.3 Assembly of ribosomal subunits…………………………………………………... 13 1.3.1 rRNA maturation and modifications…………………………………………. 13 1.3.2 Binding of ribosomal proteins and rRNA folding…………………………... 15 1.3.3 Ribosomal assembly factors………………………………………………........ 18 DEAD‐ box proteins, GTPases, Chaperones and maturation factor…….................... 19 1.4 Bacterial translation initiation…………………………………………………...... 21 1.4.1 The translation initiation region in messenger RNA………………………... 22 1.4.2 The initiator transfer RNA (itRNA)…………………………………………… 22 Transfer RNA modification……………………………................................................. 23 1.4.3 Initiation factor 1 (IF1)………………………………………………………….. 24 1.4.4 Initiation factor 2 (IF2)………………………………………………………….. 26 1.4.5 Initiation factor 3 (IF3)………………………………………………………….. 27 1.4.6 The antibiotic kasugamycin as a translation initiation inhibitor…………… 28 1.5 Bacterial translation termination and recycling………………………………….. 30 1.5.1 Class 1 release factors…………………………………………………………. 30 2. Results and discussion……………………………………………………………… 34 2.1 Paper I……………………………………………………………………………….. 34 2.2 Paper II…………………………………………….................................................... 36 2.3 Paper III…................................................................................................................... 37 3. Concluding remarks………………………………………………………………… 39 4. Acknowledgements…………………………………………………………………. 40 5. References…………………………………………………………………………...... 41 7 Abbreviations ASD anti Shine‐Dalgarno sequence A‐site aminoacyl‐tRNA binding site ASL anticodon stem‐loop CP central proturberance DASL dimethyl‐A stem‐loop DR The downstream region E‐site exit‐tRNA binding site EF‐Tu Elongation factor Tu fMet ‐tRNA fMet formylated initiator tRNA GDP guanosine diphosphate GTP guanosine triphosphate GTPase enzyme hydolyzing GTP IF1, 2, 3 initiation factor itRNA initiator tRNA H helix HSP heat shock protein LB Luria‐Bertani medium mRNA massenger RNA Nt nucleotide OB oligomer‐ binding P precursor PIC pre‐initiation complex P‐site peptidyl‐tRNA binding site PTC peptidyl transferase center RBS ribosome binding site RF1, 2, 3 release factor RRF ribosome recycling factor rRNA ribosomal RNA SD Shine‐Dalgarno sequence TIR translation initiation region tRNA transfer RNA Å Ångstöm, 1Å = 1 × 10‐10 m 8 1. Introduction 1.1 Bacterial translation process Protein synthesis, or translation of the genetic information in mRNA (messenger RNA) into amino acid sequence of proteins, is essentially the same in all kingdoms of life and takes place on the ribosome. The ribosome which is formed of two subunits (30S and 50S subunits in bacteria) contains three binding sites for tRNA molecules: the A site binds the aminoacyl‐tRNA, the P site binds the peptidyl‐tRNA and the E site binds the deacylated tRNA. Protein synthesis can be divided into four main steps, initiation, elongation, termination and recycling. During initiation, the initiation factors (IFs) facilitate the assembly of the 30S and 50S subunits on mRNAs translation initiation region (TIR) to form an active ribosomal particle and the placement of the initiator tRNAfMet in the P‐site [2, 3]. At the end of the initiation step the A site is ready to receive an aminoacyl‐tRNA molecule which is delivered by elongation factor EF‐Tu. The proper codon‐anticodon interactions stimulate the GTP‐ase activity of EF‐Tu leading to the dissociation of EF‐Tu from the complex. As a consequence the aminoacyl end of the A‐site tRNA releases and positions in the peptidyl‐transferase center (PTC) in a process known as accommodation. A peptide bond is then formed between the A‐ and P‐site tRNAs (α amino group of the aminoacyl‐tRNA attacks the carbonyl carbon of the peptidyl‐tRNA) at the peptidyl‐transferase center on the 50S subunit. Upon peptide bond synthesis, the lengthened peptidyl‐tRNA is bound to the A site, whereas the deacylated tRNA is in the P‐site. Peptide elongation is further promoted by the GTP‐dependent protein elongation factors EF‐G. The GTP hydrolysis of EF‐G promotes the translocation of peptidyl‐tRNA (carrying a peptide chain one amino acid longer) from the A‐site to the P‐site and the deacylated tRNA from the P‐site to the E‐site. Consequently, the ribosome moves down the mRNA with an empty A‐site ready to receive a new tRNA molecule, the positioning of it is promoted by the elongation factor EF‐Tu [4‐6]. Protein synthesis in bacteria terminates when a stop codon on mRNA enters the ribosomal A‐site. Release factor 1 or 2 recognizes the stop codon and subsequently catalyses the hydrolysis of peptidyl‐tRNA, releasing the nascent polypeptide from the ribosome [7]. Release factor 3 9 triggers the dissociation of release factor 1 or 2 from the A‐site [8]. After the dissociation of RF3 from the ribosome, the ribosome must be recycled into subunits for a new round of translation initiation. Ribosome recycling factor RRF, EF‐G and IF3 proteins are required to release the deacylated tRNA, mRNA and to dissociate the ribosome subunits [9, 10]. In this study we will focus in two processes: the translation initiation and termination in bacteria. 1.2 The bacterial ribosome and its subunits The ribosome is the largest and the most complex ribozyme found in nature. It consists of two subunits of unequal size, a small 30S and a large 50S subunit, assembles upon translation initiation and has a relative sedimentation rate of 70S. The amount of ribosomes is tightly regulated because making ribosomes is costly for the cell. The eubacteria Escherichia coli (E.coli) cell contains about 2,000 ribosomes at slow growth rate and this number can increase to 70,000 per cell during rapid growth [11]. Many cryo electron microscopy (cryo‐EM) studies have improved the structural knowledge of the ribosome and revealed new features such as the localization of several translation factors and a folded mRNA and the conformational changes associated with different functional states [12‐15]. In E. coli, one
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