Differential Effects of Thiopeptide and Orthosomycin Antibiotics on Translational GTPases

SummaryThe ribosome is a major target in the bacterial cell for antibiotics. Here, we dissect the effects that the thiopeptide antibiotics thiostrepton (ThS) and micrococcin (MiC) as well as the orthosomycin antibiotic evernimicin (Evn) have on translational GTPases. We demonstrate that, like ThS, MiC is a translocation inhibitor, and that the activation by MiC of the ribosome-dependent GTPase activity of EF-G is dependent on the presence of the ribosomal proteins L7/L12 as well as the G′ subdomain of EF-G. In contrast, Evn does not inhibit translocation but is a potent inhibitor of back-translocation as well as IF2-dependent 70S-initiation complex formation. Collectively, these results shed insight not only into fundamental aspects of translation but also into the unappreciated specificities of these classes of translational inhibitors

Translationale Regulation der Release-Faktor 2 Synthese

Title Page Table of Contents Abstract III Zusammenfassung IV 0\. Abbreviations 1 Chapter 1: Introduction 3 1.1 Ribosome: A protein synthesis factory 3 1.2 Translational errors and two tRNAs on the ribosome 12 1.3 Mechanism of genetic expression of RF2 protein: an autoregulatory mechanism 13 2\. Chapter 2: Materials and Methods 15 2.1 Materials 15 2.2 Buffers 18 2.3 Analytical methods 26 2.4 Working with DNA 30 2.5 Working with RNA 35 2.6 Preparative Methods 51 2.7 In vitro systems 59 2.8 Computational analysis: Secondary structure prediction of synthetic RNA and estimation of its ?° of formation 67 3\. Chapter 3: Results 68 3.1 Pre-requisites for the analysis of RF2 frameshifting mechanism 68 3.2 Characterisation of the in vitro translation system for the RF2 mRNA model 79 3.3 Analysis of the frameshift window 88 3.4 Protein synthesis termination: an effective in vitro system 91 3.5 Kinetic evaluation of the frameshifting mechanism 93 3.6 The effects of SD on the E site tRNA, termination process and frameshifting 95 3.7 Location of Shine-Dalgarno sequence. Effect on frameshifting 96 3.8/3.9 Di-peptide formation 99 3.10 Translocation efficiency 106 4\. Discussion 108 4.1 Rnase method 108 4.2 In vitro translation system 109 4.3 Di-peptide formation 114 4.4 RF2 and the release of deacylated tRNA from the E site 115 4.5 Frameshifting mechanism in translation of the RF2 mRNA 116 1\. BibliographyRibosomes translate the genetic information encoded in the mRNA with an extremely high efficiency and accuracy. In this respect, maintenance of the correct reading frame is one of the major tasks achieved by the ribosomes during the protein synthesis process. A spontaneous change in the reading frame generates truncated and usually non-functional proteins resulting in the loss of the genetic information. Normally a spontaneous frame-shift occurs approximately once in 30,000 incorporations of amino acids. However, in the case of the synthesis of the termination factor a frameshift has to occur, since the 26th codon of the RF2 mRNA is the stop codon UGA. At this stop codon a +1 frameshift is required for the RF2 synthesis, and it occurs with an efficiency of up to 100% if the RF2 concentration is low, that is with a frequency that is four orders of magnitude larger than that normally observed during protein synthesis. The presence of the internal stop codon of the RF2 mRNA is the basis of the translational feed-back regulation of the RF2 synthesis. If the concentration of RF2 is sufficiently high in the cell, RF2 recognizes the stop codon UGA on its own RF2 mRNA at the codon position 26, with the result that an oligopeptide of 25 amino acids is synthesized, released and fast degraded. However, at a low concentration of RF2 a (+1) frameshift occurs that is necessary for a complete synthesis of the RF2 protein. In this work the mechanism of this extreme frameshift event has been elucidated and resolved. Moreover, evidence is provided that an occupation of the E site with a deacylated tRNA is essential for maintaining the reading frame. We demonstrate that the internal Shine Dalgarno (SD) sequence in front of the stop codon UGA (26th codon) of the RF2 mRNA plays a critical role for the mechanisms of the RF2 feed-back regulation. When the internal UGA is at the A site, the SD sequence is separated from the peptidyl-tRNA at the P site by an extremely short spacer sequence of only two nucleotides and thus interferes with the first nucleotide of the E site codon. The result is a steric clash between the SD-antiSD of the 16S rRNA and codon-anticodon interaction, and we show that this clash triggers the release of the E-site tRNA. The resulting ribosome with only one tRNA, the peptidyl-tRNA at the P site, is a situation that never exists during elongation, where statistically always two tRNAs are on the ribosome. The short spacer forces this non- elongating ribosome to move (+1) nucleotide downstream displaying the frameshifted codon GAC for Asp at the A site. We demonstrate that the loss of the tRNA at the E site is correlated with the incorporation of Asp. However, when the SD sequence is shifted by two or six nucleotides upstream from the wild type position, the tRNA at the E-site is not released and a frameshift does not occur. The in vitro translation system developed for the analysis of the frameshift at the RF2 mRNA shows for the first time a frameshift frequency near 100% reflecting the known data in vivo. We further demonstrate that the E-site tRNA is not removed by a normal RF2 "decoding" of the stop codon UGA. Therefore, the tRNA release from the E site has to occur at a later step of a normal termination process, a fact that is neglected by current models of termination. Our data demonstrate further for the first time that a cognate tRNA at the E site is instrumental for maintaining the reading frame, and that the loss of the E site tRNA is the trigger for the enormous efficiency of frameshifting during the translational regulation of the RF2.Ribosomen übersetzen die genetische Information, die in der mRNA kodiert ist, mit extrem hoher Effizienz und Genauigkeit. In dieser Hinsicht ist die Aufrechterhaltung des korrekten Leserahmens während der Proteinsynthese eine der wesentlichen Aufgaben des Ribosoms. Ein spontaner Wechsel des Leserahmens führt zu unvollständigen und gewöhnlich nicht funktionsfähigen Proteinen und damit zu einem Verlust der genetischen Information. Normalerweise kommt eine spontane Änderung des Leserahmens einmal alle 30.000 eingebauten Aminosäuren vor. Im Fall der RF2 Synthese muss jedoch ein Leserahmenwechsel in +1 Richtung erfolgen, da das 26igste Codon das Stoppcodon UGA ist, das mit dem Leserahmenwechsel bei niedriger RF2 Konzentration umgangen wird und erst durch den Leserahmenwechsel zur vollständigen Synthese von RF2 führt. Der Leserahmenwechsel erfolgt mit einer erstaunlichen Effizienz von bis zu 100%, d.h. mit einer um vier Zehnerpotenzen größeren Häufigkeit als bei der üblichen Proteinsythese. Das interne Stoppcodon der RF2 mRNA ist die Basis der regulatorischen Rückkopplung der RF2 Synthese. Falls die RF2 Konzentration genügend hoch ist, erkennt RF2 das interne Stoppcodon an der 26igsten Codonposition seiner eigenen mRNA, mit dem Ergebnis, dass ein Oligopeptid von 25 Aminosäuren synthetisiert, vom Ribosom entlassen und schnell abgebaut wird. Falls hingegen die RF2 Konzentration niedrig ist, erfolgt der beschriebene +1 Leserahmenwechsel, was die RF2 Synthese ermöglicht. In dieser Arbeit wird der Mechanismus des extrem effizienten Leserahmen-wechsels aufgeklärt. Darüber hinaus wird nachgewiesen, dass die Besetzung der E Stelle eine Voraussetzung für die Erhaltung des Leserahmens während der Proteinsynthese ist. Wir zeigen, dass die interne Shine-Dalgarno (SD) Sequenz vor dem internen Stoppcodon UGA (26igster Codon) der RF2-mRNA von kritischer Bedeutung für den Mechanismus der Regulation der RF2 Synthese ist. Wenn das interne Stoppcodon UGA in der A Stelle angelangt ist, liegt die SD Sequenz nur mit einer extrem kurzen Spacersequenz von 2 Nukleotiden stromaufwärts von der Peptidyl-tRNA in der P Stelle und überlappt damit mit der ersten Codonposition der E-Stellen tRNA. Dieser Sachverhalt führt zu einem sterischen Konflikt zwischen SD-AnitSD der 16S rRNA und Codon-Anticodon-Wechselwirkung in der E Stelle, und dieser sterische Zusammenstoß, wie hier gezeigt wird, führt zur Entlassung der tRNA aus der E Stelle. Damit besitzt das Ribosom nur eine tRNA, nämlich die Peptidyl-tRNA in der P Stelle, eine Situation, die während der Elongation nicht vorkommt, da während der Elongation statistisch immer zwei tRNAs auf dem Ribosom befinden. Wegen des kurzen Spacers drückt die SD-AntiSD Wechselwirkung das Ribosom mit seiner einen tRNA um ein Nukleotid stromabwärts, was das +1 Codon GAC für Asp in die A Stelle einstellt. Wir zeigen, dass der Verlust der tRNA aus der E Stelle mit dem Einbau Asp Aminosäure einhergeht. Wenn jedoch die SD Sequenz um zwei oder sechs Nukleotiden stromaufwärts verschoben wird, bleibt die Entlassung der tRNA aus der E Stelle sowie ein Wechsel des Leserahmens aus (kein Einbau von Asp). Das in vitro Translationssystem, das für die Analyse der RF2-mRNA Translation entwickelt wurde, kann zum ersten mal in vitro einen Leserahmenwechsel von bis zu 100% entsprechend den in vivo Befunden nachweisen. Wir zeigen ferner, dass die E-Stellen tRNA bei einer "normalen" UGA Dekodierung durch RF2 nicht entlassen wird. Deshalb muss die E-Stellen tRNA in einem späteren Schritt der Terminationsphase entlassen werden, was in den vorherrschenden Modellen der Translations-Termination nicht berücksichtigt ist. Unsere Daten demonstrieren weiterhin zum ersten mal, dass eine tRNA in der E Stelle für die Aufrechterhaltung des Leserahmens von fundamentaler Bedeutung ist, und dass ein Verlust der E-Stellen tRNA den enorm effizienten Leserahmenwechsel während der RF2 Synthese auslöst

Switching off the mechanism for maintaining the ribosomal reading frame: translational regulation of release factor

Ribosomes translate the genetic information encoded in the mRNA with an extremely high efficiency and accuracy. In this respect, maintenance of the correct reading frame is one of the major tasks achieved by the ribosomes during the protein synthesis process. A spontaneous change in the reading frame generates truncated and usually non-functional proteins resulting in the loss of the genetic information. Normally a spontaneous frame-shift occurs approximately once in 30,000 incorporations of amino acids. However, in the case of the synthesis of the termination factor a frameshift has to occur, since the 26th codon of the RF2 mRNA is the stop codon UGA. At this stop codon a +1 frameshift is required for the RF2 synthesis, and it occurs with an efficiency of up to 100% if the RF2 concentration is low, that is with a frequency that is four orders of magnitude larger than that normally observed during protein synthesis. The presence of the internal stop codon of the RF2 mRNA is the basis of the translational feed-back regulation of the RF2 synthesis. If the concentration of RF2 is sufficiently high in the cell, RF2 recognizes the stop codon UGA on its own RF2 mRNA at the codon position 26, with the result that an oligopeptide of 25 amino acids is synthesized, released and fast degraded. However, at a low concentration of RF2 a (+1) frameshift occurs that is necessary for a complete synthesis of the RF2 protein. In this work the mechanism of this extreme frameshift event has been elucidated and resolved. Moreover, evidence is provided that an occupation of the E site with a deacylated tRNA is essential for maintaining the reading frame. We demonstrate that the internal Shine Dalgarno (SD) sequence in front of the stop codon UGA (26th codon) of the RF2 mRNA plays a critical role for the mechanisms of the RF2 feed-back regulation. When the internal UGA is at the A site, the SD sequence is separated from the peptidyl-tRNA at the P site by an extremely short spacer sequence of only two nucleotides and thus interferes with the first nucleotide of the E site codon. The result is a steric clash between the SD-antiSD of the 16S rRNA and codon-anticodon interaction, and we show that this clash triggers the release of the E-site tRNA. The resulting ribosome with only one tRNA, the peptidyl-tRNA at the P site, is a situation that never exists during elongation, where statistically always two tRNAs are on the ribosome. The short spacer forces this non-elongating ribosome to move (+1) nucleotide downstream displaying the frameshifted codon GAC for Asp at the A site. We demonstrate that the loss of the tRNA at the E site is correlated with the incorporation of Asp. However, when the SD sequence is shifted by two or six nucleotides upstream from the wild type position, the tRNA at the E-site is not released and a frameshift does not occur. The in vitro translation system developed for the analysis of the frameshift at the RF2 mRNA shows for the first time a frameshift frequency near 100% reflecting the known data in vivo. We further demonstrate that the E-site tRNA is not removed by a normal RF2 "decoding" of the stop codon UGA. Therefore, the tRNA release from the E site has to occur at a later step of a normal termination process, a fact that is neglected by current models of termination. Our data demonstrate further for the first time that a cognate tRNA at the E site is instrumental for maintaining the reading frame, and that the loss of the E site tRNA is the trigger for the enormous efficiency of frameshifting during the translational regulation of the RF2.0 Abbreviations.................................................................................................1 1.1. Ribosome: A protein synthesis factory..........................................................................3 1.1.1. Initiation..................................................................................................................5 1.1.2. Elongation...............................................................................................................6 1.1.2.1. General description..........................................................................................6 1.1.2.2. Models for the elongation cycle.......................................................................7 1.1.3. Termination.............................................................................................................8 1.1.3.1. General description..........................................................................................8 1.1.3.2. How is termination achieved?..........................................................................9 1.1.3.3. Recycling........................................................................................................10 1.2. Translational errors and two tRNAs on the ribosome..................................................12 1.3. Mechanism of genetic expression of RF2 protein: an autoregulatory mechanism......13 2.1 Materials.......................................................................................................................15 2.1.1 Chemicals and enzymes-Suppliers.........................................................................15 2.2. Buffers.........................................................................................................................18 2.2.1 Buffers and Electrophoresis solutions...................................................................18 2.2.2 Buffers for Microbiological and Molecular methods.............................................21 2.2.3 Buffers for the functional studies and ribosome preparation................................23 2.3 Analytical methods........................................................................................................26 2.3.1 Determination of ribosome and nucleic acid concentrations................................26 2.3.2 Conversion factors for the quantification of DNA and RNA..................................26 2.3.3 Radioactivity measurements...................................................................................27 2.3.4 Cold TCA precipitation for the quantitative determination of aminoacylated tRNA........................................................................................................................................27 2.3.5 Agarose gel electrophoresis of DNA and RNA......................................................28 2.3.6 Specific activity determination of labelled [32P]-tRNA..........................................29 2.3.7 Western blot of tRNA-free S-100 fraction..............................................................29 2.4 Working with DNA.......................................................................................................30 2.4.1 Preparation of E. coli competent cells for electroporation...................................30 2.4.2. Cloning strategies.................................................................................................31 2.4.3. Restriction with EcoRI and BamHI.......................................................................31 2.4.4. Digestion with alkaline phosphatase....................................................................32 2.4.5. Synthesis of dsDNA and ligation to a linearized plasmid.....................................32 2.4.6. Annealing and DNA filling reaction.....................................................................32 2.4.7. Ligation to linearized plasmid..............................................................................32 2.4.8. Transformation......................................................................................................33 2.4.9. Phenol/Chloroform extraction..............................................................................33 2.4.10. Nucleic acid Precipitation by Ethanol or Isopropanol.......................................34 2.4.11. Plasmid isolation-mini-prep................................................................................34 2.4.12. Plasmid preparation (maxi prep)........................................................................34 2.5. Working with RNA......................................................................................................35 2.5.1. Transcription.........................................................................................................35 2.5.1.1. Run-off transcription with T7 polymerase.....................................................35 2.5.1.2. PAGE purification of in vitro mRNA transcript............................................36 2.5.1.3. Separation at the single nucleotide level (sequencing gel)............................37 2.5.1.4. Gel filtration for the separation of RNA preparations from low molecular weight contaminants....................................................................................................38 2.5.1.5. List of messengers (mRNAs) constructed in this study.................................39 vii2.5.1.6. List of primers for the construction of the mRNA.........................................40 2.5.2. tRNAs.....................................................................................................................41 2.5.2.1. Analytical tRNA aminoacylation...................................................................41 2.5.2.2. Analytical enzymatic deacylation of aminoacyl-tRNA.................................42 2.5.2.3. Preparative tRNA aminoacylation and subsequent actylation.......................42 2.5.2.4. Preparative deacylation of aminoacyl-tRNA remaining in the N-acetylaminoacyl-tRNA fraction..................................................................................44 2.5.2.5. Reversed-Phase HPLC purification of aminoacyl-tRNA and acetylaminoacyl-tRNA...........................................................................................................................45 2.5.2.6. Preparation of N-formyl-methionyl-tRNAfMet (E. coli)...............................46 2.5.2.6.1. Preparation of the formyl donor..............................................................46 2.5.2.6.2. Synthesis and purification of fMet-tRNAfMet..........................................47 2.5.2.7. Isolation and purification of Asp-tRNAAsp....................................................47 2.5.2.8. Labelling of deacylated tRNA with γ-[32P]-ATP...........................................49 2.5.2.8.1. Dephosphorylation of tRNA with alkaline phosphates...........................49 2.5.2.8.2. [5’] Phosphorylation with [γ-32P]-ATP...................................................50 2.6. Preparative Methods.....................................................................................................51 2.6.1. Large-scale cultures of Escherichia coli...............................................................51 2.6.2. Isolation of 70S ribosomes from Escherichia coli................................................51 2.6.3. Preparative isolation of 30S and 50S subunits.....................................................52 2.6.4 Preparation of Re-associated 70S..........................................................................54 2.6.4.1. Quality and functionality determination of the ribosomes preparation.........55 2.6.4.2. Analytical sucrose gradient centrifugation.....................................................55 2.6.4.3. Integrity of rRNA-1D tube gel analysis.........................................................56 2.6.5. Preparation of the S-100 fraction from Escherichia coli......................................57 2.6.5.1. Preparation of S-100 tRNA-free....................................................................57 2.6.6. High Salt Wash Protein (HSWP) Preparation......................................................58 2.6.6.1. HSWP tRNA free Preparation........................................................................59 2.7. In vitro systems............................................................................................................59 2.7.1. Estimation of the functional competence of ribosome preparations.....................59 2.7.1.1. Poly(U)-dependent poly(Phe) synthesis.........................................................59 2.7.1.2. Determination of the AcPhe-tRNAPhe binding...............................................60 2.7.2. Watanabe assay: site specific binding of tRNA to ribosomes, translocation and puromycin reaction.........................................................................................................60 2.7.2.1. First step: P site binding or Pi complex formation.........................................61 2.7.2.2. Second step: A site binding and/or PRE complex formation.........................62 2.7.2.3. Third step: Translocation reaction.................................................................62 2.7.2.4. Fourth step: puromycin reaction....................................................................63 2.7.5 In vitro translation system for the RF2 model-mRNAs (translational reaction)...64 2.7.6 Di-peptide formation..............................................................................................65 2.7.7 RNase assay............................................................................................................66 2.8 Computational analysis: Secondary structure prediction of synthetic RNA and estimation of its ∆G° of formation......................................................................................67 3.1 Pre-requisites for the analysis of RF2 frameshifting mechanism.................................68 3.1.1 Development of a novel method for the detection of mRNA degradation: RNase Assay...............................................................................................................................68 3.1.2 In vitro translation system for RF2 mRNA expression...........................................70 3.1.2.1. MFold secondary structure prediction of the designed mRNAs....................71 3.1.2.2. Purity of mRNAs............................................................................................74 viii3.1.2.3. Translational control experiments with the newly designed mRNAs............75 3.1.3 Does S-100 tRNA free fraction contains RF2?......................................................76 3.1.3.1. Purity of Release Factors...............................................................................77 3.1.4. tRNA bulk minus tRNA Tyrosine (tRNAbulk - Tyr)....................................................78 3.2 Characterisation of the in vitro translation system for the RF2 mRNA model.............79 3.2.1 Binding assay.........................................................................................................79 3.2.2 Translational assays...............................................................................................81 3.2.2.1 Translation of the (UUC)12 sequence..............................................................81 3.2.2.2 Ribosomal Active Fraction..............................................................................82 3.2.2.3 Translation of the heteropolymeric part of the mRNA constructs..................84 Nitrocellulose filtration..................................................................................................85 TCA precipitation..........................................................................................................85 3.3 Analysis of the frameshift window...............................................................................88 3.4 Protein synthesis termination. An effective in vitro system.........................................91 3.5 Kinetic evaluation of the frameshifting mechanism.....................................................93 3.6 The effects of SD on the E site tRNA, termination process and frameshifting............95 3.7 Location of Shine-Dalgarno sequence. Effect on frameshifting...................................96 3.8 Di-peptide formation.....................................................................................................99 3.9.1 Di-peptide formation on non-programmed ribosomes?........................................99 3.9.2 Di-peptide formation in the presence of RF2: Pi versus POST complex.............103 3.10 Translocation efficiency............................................................................................106 4.1 RNase method.............................................................................................................108 4.2 In vitro translation system...........................................................................................109 4.3 Di-peptide formation...................................................................................................114 4.4 RF2 and the release of deacylated tRNA from the E site............................................115 4.5 Frameshifting mechanism in translation of the RF2 mRNA.......................................11

Functions and interplay of the tRNA-binding sites of the ribosome

The ribosome translates the genetic information of an mRNA molecule into a sequence of amino acids. The ribosome utilizes tRNAs to connect elements of the RNA and protein worlds during protein synthesis, i.e. an anticodon as a unit of genetic information with the corresponding amino acid as a building unit of proteins. Three tRNA-binding sites are located on the ribosome, termed the A, P and E sites. In recent years the tRNA-binding sites have been localized on the ribosome by three different techniques, small-angle neutron scattering, cryo-electron microscopy and X-ray analyses of 70 S crystals. These high-resolution glimpses into various ribosomal states together with a large body of biochemical data reveal an intricate interplay between the tRNAs and the three ribosomal binding sites, providing an explanation for the remarkable features of the ribosome, such as the ability to select the correct ternary complex aminoacyl-tRNA·EF-Tu·GTP out of more than 40 extremely similar tRNA complexes, the precise movement of the tRNA2 · mRNA complex during translocation and the maintenance of the reading frame

Shine–Dalgarno interaction prevents incorporation of noncognate amino acids at the codon following the AUG

During translation, usually only one in ≈400 misincorporations affects the function of a nascent protein, because only chemically similar near-cognate amino acids are misincorporated in place of the cognate one. The deleterious misincorporation of a chemically dissimilar noncognate amino acid during the selection process is precluded by the presence of a tRNA at the ribosomal E-site. However, the selection of first aminoacyl-tRNA, directly after initiation, occurs without an occupied E-site, i.e., when only the P-site is filled with the initiator tRNA and thus should be highly error-prone. Here, we show how bacterial ribosomes have solved this accuracy problem: In the absence of a Shine–Dalgarno (SD) sequence, the first decoding step at the A-site after initiation is extremely error-prone, even resulting in the significant incorporation of noncognate amino acids. In contrast, when a SD sequence is present, the incorporation of noncognate amino acids is not observed. This is precisely the effect that the presence of a cognate tRNA at the E-site has during the elongation phase. These findings suggest that during the initiation phase, the SD interaction functionally compensates for the lack of codon–anticodon interaction at the E-site by reducing the misincorporation of near-cognate amino acids and prevents noncognate misincorporation