Elsevier S & T

Translation Initiation: Reconstituted Systems and Biophysical Methods: Reconstituted Systems and Biophysical Methods

Lorsch, Jon

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Table of contents
  • Contentsv
  • Contributorsxiii
  • Prefacexix
  • Volume in Seriesxxi
  • Chapter 1: Transient Kinetics, Fluorescence, and FRET in Studies of Initiation of Translation in BacCover
  • 1. Introduction2
  • 2. Experimental Outline5
  • 3. Materials9
  • 3.1. Stock solutions9
  • 3.2. Reagents9
  • 4. Experimental Procedures10
  • 4.1. Preparation and fluorescence labeling of ribosomes and ribosomal subunits10
  • 4.2. Zonal centrifugation11
  • 4.3. Preparation and fluorescence labeling of initiator fMet-tRNAfMet12
  • 4.4. Preparation of fluorescence-labeled mRNA15
  • 4.5. Initiation factors15
  • 5. Rapid Kinetic Measurements20
  • 5.1. Measuring fluorescence intensities and FRET changes in stopped flow20
  • 6. Quench-Flow Measurements21
  • 7. GTPase Activity22
  • 8. Dipeptide Formation22
  • 9. Applications of the Method23
  • 10. Binding of fMet-tRNA to the 30S Subunit23
  • 11. FRET to IF323
  • 12. Nucleotide Binding to IF225
  • 13. Subunit Joining25
  • Acknowledgments26
  • References26
  • Chapter 2: Binding of mRNA to the Bacterial Translation Initiation Complex31
  • 1. Introduction32
  • 2. Experimental Procedures33
  • 2.1. Design of model mRNAs33
  • 2.2. Synthesis of model mRNAs33
  • 2.3. Deprotection34
  • 2.4. Thermal melting analysis34
  • 2.5. Labeling and purification of mRNA35
  • 2.6. Labeling schemes36
  • 2.7. Fluorophores37
  • 2.8. Isolation of the 30S subunit and protein purification37
  • 2.9. Purification of aminoacylated initiator tRNA39
  • 2.10. Initiation complex assembly39
  • 2.11. mRNA dissociation40
  • 2.12. Steady-state fluorescence measurements40
  • 2.13. Kinetic association and dissociation experiments41
  • 2.14. Determination of equilibrium binding constants42
  • Acknowledgments43
  • References43
  • Chapter 3: Real-Time Dynamics of Ribosome-Ligand Interaction by Time-Resolved Chemical Probing Metho45
  • 1. Introduction46
  • 2. General Strategy46
  • 2.1. Buffers and mixtures47
  • 2.2. Analysis of probing reactions48
  • 3. Time-Resolved Chemical Probing with DMS48
  • 3.1. Validation of time-resolved chemical probing with DMS49
  • 3.2. Procedure for chemical probing with DMS50
  • 3.3. Example of time-resolved chemical probing with DMS51
  • 4. Time-Resolved Probing with ONOOK51
  • 4.1. Validation of time-resolved probing with ONOOK51
  • 4.2. Preparation of ONOOK53
  • 4.3. Procedure for time-resolved probing with ONOOK54
  • 4.4. Example of time-resolved probing with ONOOK54
  • 5. Time-Resolved Chemical Probing with Fe(II)-EDTA54
  • 5.1. Validation of time-resolved probing with Fe(II)-EDTA56
  • 5.2. Procedure for time-resolved. probing with Fe(II)-EDTA56
  • 5.3. Example of time-resolved probing with Fe(II)-EDTA57
  • Acknowledgments58
  • References58
  • Chapter 4: Overexpression and Purification of Mammalian Mitochondrial Translational Initiation Facto59
  • 1. Introduction60
  • 2. Materials61
  • 2.1. Growth media61
  • 2.2. Common buffers61
  • 2.3. Buffers for IF2mt62
  • 2.4. Buffers for IF3mt62
  • 2.5. Reagents62
  • 2.6. Preparation of reagents63
  • 2.7. The high-performance liquid chromatography (HPLC) system63
  • 3. Assays for IF2mt and IF3mt63
  • 3.1. IF2mt: Principle63
  • 3.2. Assays of IF2mt on E. coli ribosomes63
  • 3.3. Assays of IF2mt on Bos taurus (Bovine) 55S mitochondrial ribosomes64
  • 3.4. Assays of IF2mt on Bos taurus (Bovine) 28S ribosomal subunits64
  • 3.5. IF3mt: Principle64
  • 3.6. Assay of IF3mt on E. coli ribosomes65
  • 3.7. Assay of IF3mt on bovine mitochondrial ribosomes65
  • 4. Purification of His-Tagged IF2MT65
  • 4.1. Growth of cells, induction, and preparation of cell extracts66
  • 4.2. Disruption by alumina grinding66
  • 4.3. Disruption by sonication67
  • 4.4. Purification of IF2mt: Ni-NTA step67
  • 4.5. Purification of IF2mt by HPLC68
  • 4.6. Purification of IF2mt on a gravity DEAE-sepharose column69
  • 5. Purification of His-Tagged IF3MT72
  • 5.1. Cell growth, induction, and preparation of cell extracts72
  • 5.2. Purification of IF3mt: Cell lysis72
  • 5.3. Purification of IF3mt: Ni-NTA step73
  • 5.4. Purification of IF3mt: Purification on HPLC73
  • 5.5. Purification of IF3mt: Gravity S-sepharose column73
  • References77
  • Chapter 5: In Vitro Studies of Archaeal Translational Initiation79
  • 1. Introduction80
  • 2. Preparation of S. solfataricus Cellular Extracts, Ribosomes, and Other Cellular Fractions82
  • 2.1. Growth of S. solfataricus cells82
  • 2.2. Preparation of whole cell lysates (S-30)82
  • 2.3. Preparation of S-100 fraction and purified ribosomes83
  • 2.4. Isolation of high-salt-purified ribosomes and "crude" initiation factors83
  • 2.5. Isolation of 30S and 50S ribosomal subunits84
  • 2.6. Preparation of bulk tRNA84
  • 2.7. Isolation of initiator met-tRNAi85
  • 2.8. Charging of tRNAi with methionine86
  • 3. In vitro Translation of Archaeal mRNAs at High Temperature86
  • 3.1. Translation in unfractionated cell lysates86
  • 3.2. RNA translation by salt-purified ribosomes87
  • 3.3. Critical parameters for in vitro translation88
  • 4. Ribosome/mRNA Interaction90
  • 4.1. Formation and detection of ribosome/mRNA complexes90
  • 4.2. Analysis of ribosome/mRNA interaction by "toeprinting"92
  • 5. Initiation Factors93
  • 5.1. Cloning and purification of S. solfataricus translation initiation factors93
  • 6. Functional Analyses of Recombinant S. solfataricus IFS96
  • 6.1. Reconstitution of trimeric a/eIF296
  • 6.2. In vitro translation of archaeal mRNA in the presence of increasing amounts of recombinant init97
  • 6.3. Cotranslation experiments98
  • 7. Interaction of Initiation Factors with Ribosomal Subunits99
  • 7.1. Analysis of IF/ribosome association by velocity sedimentation100
  • 7.2. Native gel assays103
  • 8. IF/tRNAiMet Interaction103
  • 8.1. Hydrolysis protection assay104
  • 8.2. Gel-retardation assay104
  • 9. Interaction of [35S]Met-tRNAiMet with Ribosomes105
  • 10. Translational G-Proteins106
  • Acknowledgments107
  • References107
  • Chapter 6: Reconstitution of Yeast Translation Initiation111
  • 1. Introduction112
  • 2. Large-Scale Lysis of S. cerevisiae Cells114
  • 3. Purification of 40S and 60S Ribosomal Subunits from S. cerevisiae115
  • 3.1. Culturing and storage of cells115
  • 3.2. Purification of 80S ribosomes116
  • 3.3. Gradient preparation116
  • 3.4. Separation of 80S ribosomes into 40S and 60S subunits117
  • 4. Ribosome Quality Analysis (Identity Gel)118
  • 4.1. Extracting ribosomal RNA119
  • 4.2. Gel analysis119
  • 5. Purification of His-Tagged eIF2 from S. cerevisiae120
  • 6. Purification of His-Tagged eIF3 from S. cerevisiae122
  • 7. Overexpression and Purification of Yeast eIF1, eIF1A, and eIF5 from E. coli124
  • 8. Overexpression and Purification of Yeast eIF1A in E. coli125
  • 9. Overexpression and Purification of Yeast eIF5B in E. coli127
  • 10. Purification of Yeast Methionyl-tRNA Synthetase (YMETRS) from S. cerevisiae128
  • 11. RNA Synthesis and Purification129
  • 11.1. Gel purification of transcription products130
  • 11.2. Yeast initiator tRNA130
  • 12. Charging tRNAiMet with Methionine131
  • 12.1. Limiting charging reaction132
  • 12.2. Stoichiometric charging reaction133
  • 13. Filter Binding Assay to Monitor Ternary Complex Formation134
  • 14. Benchtop eIF2 GTPase Assay135
  • 14.1. Gel preparation135
  • 14.2. Experimental setup136
  • 14.3. Separation of GTPgamma[32P] and 32Pi136
  • 15. 43S/80S Complex Gel Shift Assay137
  • 15.1. Preparing an acrylamide gel for the 43S/80S gel shift assay137
  • 15.2. Preparing 43S gel shift reactions139
  • 15.3. Preparing 80S gel shift reactions140
  • 15.4. Running 43S/80S gels140
  • 15.5. Disassembly and analysis of gels141
  • 15.6. Testing 40S ribosomes141
  • 16. Solutions142
  • Acknowledgments143
  • References144
  • Chapter 7: Assembly and Analysis of Eukaryotic Translation Initiation Complexes147
  • 1. Introduction148
  • 2. Chemicals, Enzymes, and Biological Materials149
  • 3. Purification of 40S and 60S Ribosomal Subunits151
  • 3.1. Buffers151
  • 4. Purification of Native eIF3 and eIF4F152
  • 4.1. Buffers152
  • 5. Purification of Native eIF2, eIF5, and eIF5B156
  • 5.1. Buffers156
  • 6. Expression in E. coli and Purification of Recombinant eIF1, eIF1A, eIF4A, eIF4B, eIF4G, eIF5, and157
  • 6.1. Buffers157
  • 7. Preparation and Aminoacylation of Initiator tRNA159
  • 7.1. Buffers159
  • 7.2. In vitro transcription of initiator tRNA159
  • 7.3. Purification of methionyl-tRNA synthetase161
  • 7.4. Aminoacylation of tRNAiMet161
  • 8. Assembly of 48S and 80S Initiation Complexes on beta-Globin mRNA162
  • 8.1. Buffer162
  • 9. Resolution of Translation Initiation Complexes by Sucrose Density Gradient Centrifugation163
  • 9.1. Buffer163
  • 10. Toe Printing Analysis of Ribosomal’Complexes164
  • 11. Site-Directed UV Cross-Linking of mRNA in Initiation Complexes166
  • 11.1. Buffers166
  • 11.2. Identification of cross-linked initiation factors and ribosomal proteins169
  • 11.3. Identification of cross-linked nucleotides in 18S rRNA170
  • 12. Directed Hydroxyl Radical Probing to Localize Initiation Factors on the 40S Subunit171
  • 12.1. Buffers171
  • Acknowledgments174
  • References175
  • Chapter 8: Reconstitution of Mammalian 48S.Ribosomal Translation Initiation Complex179
  • 1. Introduction180
  • 2. Reagents182
  • 3. Buffers182
  • 3.1. Met-tRNAi preparation182
  • 3.2. Ribosome preparation183
  • 3.3. Purification of translation initiation factors from rabbit reticulocyte lysates183
  • 4. General Methods184
  • 4.1. Preparation of initiator methionyl-tRNA (Met-tRNAi)184
  • 4.2. Preparation of ribosomal subunits185
  • 4.3. Preparation of AUG trinucleotide codon and mRNA186
  • 4.4. Purification of translation initiation factors from rabbit reticulocyte lysates188
  • 4.5. Purification of initiation factors195
  • 4.6. Further purification of eIF1A from the 1 M KCl phosphocellulose eluate198
  • 4.7. Purification of eIF4F from rabbit reticulocyte lysate199
  • 4.8. Purification of recombinant translation initiation factors eIF5, eIF1, eIF1A, and eIF4B200
  • 5. Reconstitution of the 48S Ribosomal Complex203
  • 5.1. mRNA binding by the 43S preinitiation complex203
  • 5.2. Primer extension assays204
  • 6. Remarks206
  • 6.1. eIF2-mediated ternary complex assay206
  • 6.2. Assays for initiation factors206
  • 6.3. Binding of mRNA to the 43S preinitiation complex206
  • 6.4. eIF5B-/mediated 80S initiation complex formation207
  • Acknowledgments207
  • References207
  • Chapter 9: Biophysical Approach to Studies of Cap-eIF4E Interaction by Synthetic Cap Analogs209
  • 1. Introduction210
  • 2. Syntheses and Purification of Cap Analogs212
  • 2.1. Synthesis of P1-guanosine-5' P3- (7-methylguanosine-5') triphosphate (m7GpppG)213
  • 2.2. Synthesis of P1-guanosine-5' P3-(N2,N2,7-trimethyguanosine-5') triphosphate (m32,2,7GpppG)215
  • 3. Binding of the Cap Analogs to eIF4E by Fluorescence Titration Experiments218
  • 3.1. Thermodynamic and apparent association constant218
  • 3.2. Preparation of the samples220
  • 3.3. Experimental conditions of fluorescence titration221
  • 3.4. Titration assay222
  • 3.5. Corrections of fluorescence raw data222
  • 3.6. Fluorescence data analysis224
  • 3.7. Statistical analysis225
  • 3.8. Activity of the nonenzymatic eIF4E protein226
  • 3.9. Incorrectness of data linearization231
  • 4. Application of Microcalorimetry to Studying eIF4E-Cap Interaction232
  • 4.1. Principles of microcalorimetry experiments232
  • 4.2. Experimental conditions of calorimetric measurements233
  • 4.3. Modified ‘‘single injection’’ experiment233
  • 4.4. Calorimetric data treatment233
  • 4.5. Calorimetric data analysis234
  • 5. Stopped-Flow Fluorescence Studies of Binding Cap Analogs to eIF4E235
  • 5.1. Principles of stopped-flow experiments235
  • 5.2. Data analysis of ligand-binding kinetics236
  • 5.3. Practical realization of fluorescence stopped-flow experiments239
  • 5.4. Example of human eIF4E-m7GpppG association kinetics240
  • Acknowledgments242
  • References242
  • Chapter 10: Biophysical Studies of the Translation Initiation Pathway with Immobilized mRNA AnalogsCover
  • 1. Introduction248
  • 2. Generation of RNAsCover
  • 2.1. Modification and immobilization of RNAsCover
  • 2.2. The use of immobilized RNAs in the BIAcore system252
  • 2.3. The use of immobilized RNAs for atomic force microscopy255
  • 3. Protocol 1: 3'-End Biotinylation of a Transcript by Use of Poly (A) Polymerase260
  • 4. Protocol 2: Introduction of Aldehyde Groups by Oxidation of the mRNA 3'-End261
  • 5. Protocol 3: Thiolation at the mRNA 5'-End by Use of Polynucleotide Kinase262
  • Acknowledgments263
  • References263
  • Chapter 11: Protection-Based Assays to Measure Aminoacyl-tRNA Binding to Translation Initiation FactCover
  • 1. Introduction266
  • 2. Purification of Overproduced Initiator tRNACover
  • 2.1. Buffers268
  • 2.2. Protocols268
  • 3. Preparation of Methionylated tRNA270
  • 3.1. Buffers270
  • 3.2. Protocols270
  • 3.3. Reagents271
  • 3.4. Analytical assay272
  • 3.5. Preparative aminoacylation272
  • 4. Protection Assay273
  • 4.1. Buffers and reagents276
  • 4.2. Protocols276
  • 4.3. Data processing277
  • 5. Conclusion279
  • Acknowledgments279
  • References279
  • Chapter 12: NMR Methods for Studying Protein-Protein Interactions Involved in Translation InitiationCover
  • 1. IntroductionCover
  • 2. Types of Interactions in Translation from the Perspective of KD and Lifetime of the ComplexCover
  • 2.1. Overview285
  • 2.2. Types of interactions from the perspective of translation initiation288
  • 2.3. Random vs. ordered assembly of multisubunit complexes289
  • 2.4. Specific vs. nonspecific interactions289
  • 3. NMR Methods for Studying Protein Interactions in Translation289
  • 3.1. Overview289
  • 3.2. Protein size and NMR292
  • 3.3. Exchange regimes in NMR293
  • 3.4. Applications of NMR for studying protein interactions298
  • 3.5. Interactions in translation from an NMR perspective300
  • 3.6. Applications of NMR for studying protein interactions in translation300
  • 4. Descriptions of NMR Methods310
  • 4.1. Methods for backbone assignments310
  • 4.2. Methods for side-chain assignments313
  • 4.3. Methods for structure determination314
  • 4.4. Uniform isotope labeling316
  • 4.5. Specific labeling schemes (nonuniform labeling)317
  • 4.6. Segmental labeling318
  • 5. Special Cases319
  • 5.1. Interdomain interactions and orientations319
  • 5.2. Nonspecific vs specific binding320
  • 5.3. Unstable proteins or complexes320
  • 6. Summary321
  • Acknowledgments325
  • References325
  • Chapter 13: Structural Methods for Studying IRES FunctionCover
  • 1. Introduction334
  • 1.1. Structural information does not come only from high-resolution techniques335
  • 1.2. A roadmap to answer specific questions335
  • 2. Methods337
  • 2.1. Solution hydroxyl radical probing337
  • 2.2. End labeling protocols (for large RNA molecules, 150 to 400nt)344
  • 2.3. Sequencing ladders346
  • 2.4. RNase T1 structure probing347
  • 2.5. Mutagenesis/Native gels350
  • 2.6. Sedimentation velocity analytical ultracentrifugation356
  • 2.7. Crystal engineering360
  • 2.8. A 96-well tray-based method to rapidly screen many RNAs for crystallization364
  • 3. Conclusions367
  • References368
  • Chapter 14: Biophysical and Biochemical Investigations of dsRNA-Activated Kinase PKR373
  • 1. Introduction374
  • 2. Expression and Purification of PKR376
  • 3. RNA Synthesis379
  • 4. Phosphorylation Assays380
  • 5. Measuring RNA-Protein Stabilities383
  • 6. Monitoring the Association State of PKR386
  • 7. NMR Spectroscopy388
  • 8. In Vitro Translation Assays392
  • 9. Conclusions393
  • References
  • Chapter 15: Expression and Purification of Recombinant Wheat Translation Initiation Factors eIF1, eI397
  • 1. Introduction398
  • 2. Materials399
  • 3. Procedures399
  • 3.1. Cloning of cDNAs for wheat initiation factors399
  • 3.2. Growth of E. coli and expression of initiation factors400
  • 3.3. Lysis of E. coli cells401
  • 3.4. General protein purification procedures401
  • 3.5. Production of rabbit antibodies403
  • 3.6. Purification of wheat eIF1403
  • 3.7. Purification of wheat eIF1A404
  • 3.8. Purification of wheat eIF4A404
  • 3.9. Purification of wheat eIF4B404
  • 3.10. Purification of wheat eIF4F or eIF(iso)4F405
  • 3.11. Purification of eIF4G or eIF(iso)4G405
  • 3.12. Purification of eIF4E or eIF(iso)4E406
  • 3.13. Purification of wheat eIF5406
  • 4. Results406
  • 5. Conclusions407
  • Acknowledgments408
  • References408
  • Chapter 16: In Vitro Reconstitution and Biochemical Characterization of Translation Initiation by In409
  • 1. Introduction410
  • 2. Chemicals, Enzymes, and Biological Materials411
  • 3. Plasmids413
  • 4. Purification of Factors and Ribosomes414
  • 4.1. Buffers414
  • 5. Preparation of IRES-Containing mRNA and Aminoacylated Initiator tRNA415
  • 6. Strategies for Identification of Viral IRESs416
  • 7. Assays for 48S Complex Formation in Cell-Free Extracts: Sucrose Density Gradient Centrifugation417
  • 7.1. Buffers417
  • 8. Assays for 48S Complex Formation in Cell-Free Extracts: Toe Printing418
  • 8.1. Buffers418
  • 9. Identification of the Minimum Set of Factors Required for 48S Complex Formation421
  • 10. Reconstitution of 48S Complex Formation on Type 2 Picornavirus IRESs423
  • 10.1. Buffer423
  • 11. Toe Printing to Map Stable Interactions of the IRES with Components of the Translation Apparatus424
  • 12. Mapping Interactions of the IRES with Components of the Translation Apparatus by Chemical and En427
  • 12.1. Buffers427
  • 13. Directed Hydroxyl Radical Cleavage432
  • 13.1. Buffers432
  • Acknowledgments436
  • References436
  • Author Index441
  • Subject Index455
Book details
  • Vendor Elsevier S & T
  • SKU 9780123739698
  • ISBN-13 9780080553184
  • Author Lorsch, Jon
  • Category Science
  • Subject Microbiology

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For over fifty years the Methods in Enzymology series has been the critically aclaimed laboratory standard and one of the most respected publications in the field of biochemistry. The highly relevant material makes it an essential publication for researchers in all fields of life and related sciences. This volume, the second of three on the topic of Translation Initiation includes articles written by leaders in the field.