Translation Initiation: Extract Systems and Molecular Genetics

II......Page 1
V-XI......Page 2
XIII-XVII......Page 9
XIX......Page 14
XXI-XLVI......Page 15
Transient Kinetics, Fluorescence, and FRET in Studies of Initiation of Translation in Bacteria......Page 41
Introduction......Page 42
Experimental Outline......Page 45
Reagents......Page 49
Preparation and fluorescence labeling of ribosomes and ribosomal subunits......Page 50
Zonal centrifugation......Page 51
Preparation and fluorescence labeling of initiator fMet-tRNAfMet......Page 52
Initiation factors......Page 55
IF1-dependent stimulation of fMet-tRNAfMet binding to the 30S subunit......Page 59
Measuring fluorescence intensities and FRET changes in stopped flow......Page 60
Quench-Flow Measurements......Page 61
Dipeptide Formation......Page 62
FRET to IF3......Page 63
Subunit Joining......Page 65
References......Page 66
2......Page 71
Biophysical and Biochemical Investigations of dsRNA-Activated Kinase PKR......Page 406
Introduction......Page 72
Syntheses and Purification of Cap Analogs......Page 73
Lysis of E. coli cells......Page 74
Labeling and purification of mRNA......Page 75
Labeling schemes......Page 76
Applications of NMR for studying protein interactions......Page 77
Initiation complex assembly......Page 79
Steady-state fluorescence measurements......Page 80
Kinetic association and dissociation experiments......Page 81
Determination of equilibrium binding constants......Page 82
Acknowledgments......Page 83
References......Page 98
Real-Time Dynamics of Ribosome-Ligand Interaction by Time-Resolved Chemical Probing.Methods......Page 85
General Strategy......Page 86
Buffers and mixtures......Page 87
Time-Resolved Chemical Probing with DMS......Page 88
Validation of time-resolved chemical probing with DMS......Page 89
Procedure for chemical probing with DMS......Page 90
In vitro transcription of initiator tRNA......Page 197
Validation of time-resolved probing with ONOOK......Page 91
Preparation of ONOOK......Page 93
Time-Resolved Chemical Probing with Fe(II)-EDTA......Page 94
Buffer......Page 200
Purification of eIF4F from rabbit reticulocyte lysate......Page 236
Procedure for time-resolved probing with Fe(II)-EDTA......Page 96
Example of time-resolved probing with Fe(II)-EDTA......Page 97
Overexpression and Purification of Mammalian Mitochondrial Translational Initiation Factor 2 and Initiation Factor 3......Page 99
Introduction......Page 100
Overview......Page 101
Overview......Page 324
End labeling protocols (for large RNA molecules, 150 to 400nt)......Page 102
Titration assay......Page 103
IF3mt: Principle......Page 104
Assay of IF3mt on bovine mitochondrial ribosomes......Page 105
Ribosome preparation......Page 190
Disruption by alumina grinding......Page 106
Purification of IF2mt: Ni-NTA step......Page 107
Purification of IF2mt by HPLC......Page 108
Purification of IF2mt on a gravity DEAE-sepharose column......Page 109
Purification of IF3mt: Cell lysis......Page 112
Purification of IF3mt: Gravity S-sepharose column......Page 113
References......Page 315
In Vitro Studies of Archaeal Translational Initiation......Page 119
Introduction......Page 120
Structural information does not come only from high-resolution techniques......Page 122
Isolation of high-salt-purified ribosomes and "crude" initiation factors......Page 123
Preparation of bulk tRNA......Page 124
Slow exchange......Page 328
Translation in unfractionated cell lysates......Page 126
RNA translation by salt-purified ribosomes......Page 127
Critical parameters for in’vitro translation......Page 128
Formation and detection of ribosome/mRNA complexes......Page 130
Analysis of ribosome/mRNA interaction by "toeprinting"......Page 132
Stopped-Flow Fluorescence Studies of Binding Cap Analogs to eIF4E......Page 272
Cloning and purification of S. solfataricus translation initiation factors......Page 133
Reconstitution of trimeric a/eIF2......Page 136
In vitro translation of archaeal mRNA in the presence of increasing amounts of recombinant initiation factors......Page 137
Principles of stopped-flow experiments......Page 138
Interaction of Initiation Factors with Ribosomal Subunits......Page 139
Analysis of IF/ribosome association by velocity sedimentation......Page 140
IF/tRNAiMet Interaction......Page 143
Example of human eIF4E-m7GpppG association kinetics......Page 277
Gel-retardation assay......Page 144
Interaction of [35S]Met-tRNAiMet with Ribosomes......Page 145
Translational G-Proteins......Page 146
References......Page 147
Reconstitution of Yeast Translation Initiation......Page 150
Introduction......Page 151
Large-Scale Lysis of S. cerevisiae Cells......Page 153
Culturing and storage of cells......Page 154
Gradient preparation......Page 155
Separation of 80S ribosomes into 40S and 60S subunits......Page 156
Ribosome Quality Analysis (Identity Gel)......Page 157
Gel analysis......Page 158
Purification of His-Tagged eIF2 from S. cerevisiae......Page 159
Purification of His-Tagged eIF3 from S. cerevisiae......Page 161
Overexpression and Purification of Yeast eIF1, eIF1A, and eIF5 from E. coli......Page 163
Overexpression and Purification of Yeast eIF1A in E. coli......Page 164
Overexpression and Purification of Yeast eIF5B in E. coli......Page 166
Purification of Yeast Methionyl-tRNA Synthetase (YMETRS) from S. cerevisiae......Page 167
RNA Synthesis and Purification......Page 168
Purification of wheat eIF1A......Page 169
Protocols......Page 312
Mapping Interactions of the IRES with Components of the Translation Apparatus by Chemical and Enzymatic Footprinting......Page 170
Limiting charging reaction......Page 171
Stoichiometric charging reaction......Page 172
Filter Binding Assay to Monitor Ternary Complex Formation......Page 173
Gel preparation......Page 174
Separation of GTPgamma[32P] and 32Pi......Page 175
Preparing an acrylamide gel for the 43S/80S gel shift assay......Page 176
Preparing 43S gel shift reactions......Page 178
Running 43S/80S gels......Page 179
Testing 40S ribosomes......Page 180
Solutions......Page 181
Acknowledgments......Page 182
References......Page 183
Assembly and Analysis of Eukaryotic Translation Initiation Complexes......Page 185
Introduction......Page 247
Chemicals, Enzymes, and Biological Materials......Page 187
Buffers......Page 189
Buffers......Page 194
General protein purification procedures......Page 195
Aminoacylation of tRNAiMet......Page 199
Activity of the nonenzymatic eIF4E protein......Page 201
Toe Printing Analysis of Ribosomal Complexes......Page 202
Buffers......Page 204
Principles of microcalorimetry experiments......Page 269
Identification of cross-linked nucleotides in 18S rRNA......Page 208
Buffers......Page 209
Acknowledgments......Page 212
References......Page 213
Reconstitution of Mammalian 48S Ribosomal Translation Initiation Complex......Page 216
Introduction......Page 217
Buffers......Page 219
Purification of initiation factors......Page 220
Preparation of Met-tRNAi......Page 221
Overview......Page 371
Preparation of ribosomal subunits......Page 222
Synthesis of AUG......Page 223
Synthesis of capped mRNA......Page 224
Assay of eIF2 activity......Page 225
Initiation factor requirement for ribosomal complex formation vis-agrave-vis Mg2+ concentration......Page 226
Assay of eIF1 activity......Page 228
Method B......Page 229
Large-scale isolation of the 40S initiation complex......Page 230
Method B......Page 231
Initial separation of the initiation factors......Page 232
Purification of eIF2, eIF3, and eIF5 from the 700-mM KCl phosphocellulose eluate......Page 233
Further purification of eIF3......Page 234
Purification of eIF4A......Page 235
Recombinant eIF5 protein......Page 237
Recombinant eIF1 protein......Page 238
Recombinant eIF4B protein......Page 239
mRNA binding by the 43S preinitiation complex......Page 240
Primer extension assays......Page 241
Binding of mRNA to the 43S preinitiation complex......Page 243
References......Page 244
Biophysical Approach to Studies of Cap-eIF4E Interaction by Synthetic Cap Analogs......Page 246
Synthesis of P1-guanosine-5' P3-(N2,N2,7-trimethyguanosine-5') triphosphate (m3227GpppG)......Page 252
RNA Synthesis......Page 412
Preparation of the samples......Page 257
Corrections of fluorescence raw data......Page 259
Fluorescence data analysis......Page 261
Statistical analysis......Page 262
Incorrectness of data linearization......Page 268
Calorimetric data treatment......Page 270
Calorimetric data analysis......Page 271
Data analysis of ligand-binding kinetics......Page 273
Practical realization of fluorescence stopped-flow experiments......Page 276
References......Page 279
Biophysical Studies of the Translation Initiation Pathway with Immobilized mRNA Analogs......Page 283
Introduction......Page 284
End labeling with biotinylated residues......Page 286
Methods and procedures......Page 374
The use of immobilized RNAs in the BIAcore system......Page 288
The use of immobilized RNAs for atomic force microscopy......Page 291
Binding of the RNA construct to a gold-coated AFM slide......Page 293
Collecting AFM force spectroscopy data......Page 294
Analysis of AFM force spectroscopy data......Page 295
Investigating helicase activity by use of AFM RNA force spectroscopy......Page 296
Protocol 2: Introduction of Aldehyde Groups by Oxidation of the mRNA 3'-End......Page 297
Protocol 3: Thiolation at the mRNA 5'-End by Use of Polynucleotide Kinase......Page 298
References......Page 299
Protection-Based Assays to Measure Aminoacyl-tRNA Binding to Translation Initiation Factors......Page 301
Introduction......Page 302
Materials......Page 303
Protocols......Page 304
Notes......Page 305
Protocols......Page 306
Reagents......Page 307
Notes......Page 308
Protection Assay......Page 309
Data processing......Page 313
12......Page 318
Structural Methods for Studying IRES Function......Page 367
Introduction......Page 319
Types of Interactions in Translation from.the Perspective of KD and Lifetime of the Complex......Page 320
Types of interactions from the perspective of translation initiation......Page 323
Protein size and NMR......Page 327
Equilibrium between free protein and complex......Page 330
Additional considerations and special cases......Page 332
Testing for binding......Page 335
Controls......Page 336
False-negative results, or can NMR miss interactions?......Page 337
KD of the interaction......Page 338
Mapping of interaction surfaces......Page 340
Intermolecular NOEs......Page 341
Cross-saturation......Page 342
Paramagnetic labels......Page 343
Relaxation......Page 344
Methods for backbone assignments......Page 345
Methods for side-chain assignments......Page 348
Methods for structure determination......Page 349
Uniform isotope labeling......Page 351
Specific labeling schemes (nonuniform labeling)......Page 352
Segmental labeling......Page 353
Interdomain interactions and orientations......Page 354
Unstable proteins or complexes......Page 355
Summary......Page 356
References......Page 360
A roadmap to answer specific questions......Page 369
Materials......Page 373
Quantitative......Page 375
Notes and hints......Page 377
5′ End labeling: Methods and procedures......Page 378
3′ End labeling: Methods and procedures......Page 379
RNase U2: Methods and procedures......Page 380
Overview......Page 381
Methods and procedures......Page 383
Overview......Page 384
Methods and procedures......Page 388
Notes and hints......Page 389
Overview......Page 390
Methods and procedures......Page 391
Notes and hints......Page 393
Overview......Page 394
Methods and procedures......Page 395
Overview......Page 398
Methods and procedures......Page 399
Notes and hints......Page 400
Conclusions......Page 401
References......Page 402
Expression and Purification of PKR......Page 409
Phosphorylation Assays......Page 413
Measuring RNA-Protein Stabilities......Page 416
Monitoring the Association State of PKR......Page 419
NMR Spectroscopy......Page 421
In Vitro Translation Assays......Page 425
Conclusions......Page 426
References......Page 427
Expression and Purification of Recombinant Wheat Translation Initiation Factors EIF1, EIF1A, EIF4A, EIF4B, EIF4F, EIF(iso)4F, and EIF5......Page 430
Cloning of cDNAs for wheat initiation factors......Page 432
Growth of E. coli and expression of initiation factors......Page 433
Purification of wheat eIF1......Page 436
Purification of wheat eIF4B......Page 437
Purification of eIF4G or eIF(iso)4G......Page 438
Results......Page 439
Conclusions......Page 440
References......Page 441
In Vitro Reconstitution and Biochemical Characterization of Translation Initiation by Internal Ribosomal Entry......Page 442
Introduction......Page 443
Chemicals, Enzymes, and Biological Materials......Page 444
Plasmids......Page 446
Buffers......Page 447
Preparation of IRES-Containing mRNA and Aminoacylated Initiator tRNA......Page 448
Strategies for Identification of Viral IRESs......Page 449
Buffers......Page 450
Buffers......Page 451
Identification of the Minimum Set of Factors Required for 48S Complex Formation......Page 454
Buffer......Page 456
Toe Printing to Map Stable Interactions of the IRES with Components of the Translation Apparatus......Page 457
Buffers......Page 460
Buffers......Page 465
Acknowledgments......Page 469
Author Index......Page 473
Subject Index......Page 487