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

Márquez, V. (2003). Switching off the mechanism for maintaining the ribosomal reading frame: translational regulation of release factor. PhD Thesis, Freie Universität, Berlin.

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Datensatz-Permalink: https://hdl.handle.net/11858/00-001M-0000-0010-8A30-4 Versions-Permalink: https://hdl.handle.net/11858/00-001M-0000-0010-8A31-2
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 Urheber:
Márquez, Viter1, Autor
Affiliations:
1Max Planck Society, ou_persistent13              

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 Zusammenfassung: 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.

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Sprache(n): eng - English
 Datum: 2003-06-02
 Publikationsstatus: Angenommen
 Seiten: -
 Ort, Verlag, Ausgabe: Berlin : Freie Universität
 Inhaltsverzeichnis: 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.......................................116
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 Identifikatoren: eDoc: 194835
 Art des Abschluß: Doktorarbeit

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