Translation Initiation: Reconstituted Systems and Biophysical Methods

Transient Kinetics, Fluorescence, and FRET in Studies of Initiation of Translation in Bacteria......Page 1
Introduction......Page 2
Experimental Outline......Page 5
Reagents......Page 9
Preparation and fluorescence labeling of ribosomes and ribosomal subunits......Page 10
Zonal centrifugation......Page 11
Preparation and fluorescence labeling of initiator fMet-tRNAfMet......Page 12
Initiation factors......Page 15
IF1-dependent stimulation of fMet-tRNAfMet binding to the 30S subunit......Page 19
Measuring fluorescence intensities and FRET changes in stopped flow......Page 20
Quench-Flow Measurements......Page 21
Dipeptide Formation......Page 22
FRET to IF3......Page 23
Subunit Joining......Page 25
References......Page 26
Binding of mRNA to the Bacterial Translation Initiation Complex......Page 31
Introduction......Page 32
Synthesis of model mRNAs......Page 33
Thermal melting analysis......Page 34
Labeling and purification of mRNA......Page 35
Labeling schemes......Page 36
Isolation of the 30S subunit and protein purification......Page 37
Initiation complex assembly......Page 39
Steady-state fluorescence measurements......Page 40
Kinetic association and dissociation experiments......Page 41
Determination of equilibrium binding constants......Page 42
References......Page 43
Real-Time Dynamics of Ribosome-Ligand Interaction by Time-Resolved Chemical Probing.Methods......Page 45
General Strategy......Page 46
Buffers and mixtures......Page 47
Time-Resolved Chemical Probing with DMS......Page 48
Validation of time-resolved chemical probing with DMS......Page 49
Procedure for chemical probing with DMS......Page 50
Validation of time-resolved probing with ONOOK......Page 51
Preparation of ONOOK......Page 53
Time-Resolved Chemical Probing with Fe(II)-EDTA......Page 54
Procedure for time-resolved probing with Fe(II)-EDTA......Page 56
Example of time-resolved probing with Fe(II)-EDTA......Page 57
References......Page 58
Overexpression and Purification of Mammalian Mitochondrial Translational Initiation Factor 2 and Initiation Factor 3......Page 59
Introduction......Page 60
Common buffers......Page 61
Reagents......Page 62
Assays of IF2mt on E. coli ribosomes......Page 63
IF3mt: Principle......Page 64
Purification of His-Tagged IF2MT......Page 65
Disruption by alumina grinding......Page 66
Purification of IF2mt: Ni-NTA step......Page 67
Purification of IF2mt by HPLC......Page 68
Purification of IF2mt on a gravity DEAE-sepharose column......Page 69
Purification of IF3mt: Cell lysis......Page 72
Purification of IF3mt: Gravity S-sepharose column......Page 73
References......Page 77
In Vitro Studies of Archaeal Translational Initiation......Page 79
Introduction......Page 80
Preparation of whole cell lysates (S-30)......Page 82
Isolation of high-salt-purified ribosomes and "crude" initiation factors......Page 83
Preparation of bulk tRNA......Page 84
Isolation of initiator met-tRNAi......Page 85
Translation in unfractionated cell lysates......Page 86
RNA translation by salt-purified ribosomes......Page 87
Critical parameters for in’vitro translation......Page 88
Formation and detection of ribosome/mRNA complexes......Page 90
Analysis of ribosome/mRNA interaction by "toeprinting"......Page 92
Cloning and purification of S. solfataricus translation initiation factors......Page 93
Reconstitution of trimeric a/eIF2......Page 96
In vitro translation of archaeal mRNA in the presence of increasing amounts of recombinant initiation factors......Page 97
Cotranslation experiments......Page 98
Interaction of Initiation Factors with Ribosomal Subunits......Page 99
Analysis of IF/ribosome association by velocity sedimentation......Page 100
IF/tRNAiMet Interaction......Page 103
Gel-retardation assay......Page 104
Interaction of [35S]Met-tRNAiMet with Ribosomes......Page 105
Translational G-Proteins......Page 106
References......Page 107
Reconstitution of Yeast Translation Initiation......Page 110
Introduction......Page 111
Large-Scale Lysis of S. cerevisiae Cells......Page 113
Culturing and storage of cells......Page 114
Gradient preparation......Page 115
Separation of 80S ribosomes into 40S and 60S subunits......Page 116
Ribosome Quality Analysis (Identity Gel)......Page 117
Gel analysis......Page 118
Purification of His-Tagged eIF2 from S. cerevisiae......Page 119
Purification of His-Tagged eIF3 from S. cerevisiae......Page 121
Overexpression and Purification of Yeast eIF1, eIF1A, and eIF5 from E. coli......Page 123
Overexpression and Purification of Yeast eIF1A in E. coli......Page 124
Overexpression and Purification of Yeast eIF5B in E. coli......Page 126
Purification of Yeast Methionyl-tRNA Synthetase (YMETRS) from S. cerevisiae......Page 127
RNA Synthesis and Purification......Page 128
Yeast initiator tRNA......Page 129
Charging tRNAiMet with Methionine......Page 130
Limiting charging reaction......Page 131
Stoichiometric charging reaction......Page 132
Filter Binding Assay to Monitor Ternary Complex Formation......Page 133
Gel preparation......Page 134
Separation of GTPgamma[32P] and 32Pi......Page 135
Preparing an acrylamide gel for the 43S/80S gel shift assay......Page 136
Preparing 43S gel shift reactions......Page 138
Running 43S/80S gels......Page 139
Testing 40S ribosomes......Page 140
Solutions......Page 141
Acknowledgments......Page 142
References......Page 143
Assembly and Analysis of Eukaryotic Translation Initiation Complexes......Page 145
Introduction......Page 146
Chemicals, Enzymes, and Biological Materials......Page 147
Buffers......Page 149
Buffers......Page 150
Buffers......Page 154
Buffers......Page 155
In vitro transcription of initiator tRNA......Page 157
Aminoacylation of tRNAiMet......Page 159
Buffer......Page 160
Buffer......Page 161
Toe Printing Analysis of Ribosomal Complexes......Page 162
Buffers......Page 164
Identification of cross-linked initiation factors and ribosomal proteins......Page 167
Identification of cross-linked nucleotides in 18S rRNA......Page 168
Buffers......Page 169
Acknowledgments......Page 172
References......Page 173
Reconstitution of Mammalian 48S Ribosomal Translation Initiation Complex......Page 176
Introduction......Page 177
Met-tRNAi preparation......Page 179
Purification of initiation factors......Page 180
Preparation of Met-tRNAi......Page 181
Preparation of ribosomal subunits......Page 182
Synthesis of AUG......Page 183
Synthesis of capped mRNA......Page 184
Assay of eIF2 activity......Page 185
Initiation factor requirement for ribosomal complex formation vis-agrave-vis Mg2+ concentration......Page 186
Assay of eIF1 activity......Page 188
Method B......Page 189
Large-scale isolation of the 40S initiation complex......Page 190
Method B......Page 191
Initial separation of the initiation factors......Page 192
Purification of eIF2, eIF3, and eIF5 from the 700-mM KCl phosphocellulose eluate......Page 193
Further purification of eIF3......Page 194
Purification of eIF4A......Page 195
Purification of eIF4F from rabbit reticulocyte lysate......Page 196
Recombinant eIF5 protein......Page 197
Recombinant eIF1 protein......Page 198
Recombinant eIF4B protein......Page 199
mRNA binding by the 43S preinitiation complex......Page 200
Primer extension assays......Page 201
Binding of mRNA to the 43S preinitiation complex......Page 203
References......Page 204
Biophysical Approach to Studies of Cap-eIF4E Interaction by Synthetic Cap Analogs......Page 206
Introduction......Page 207
Syntheses and Purification of Cap Analogs......Page 209
Synthesis of P1-guanosine-5' P3- (7-methylguanosine-5') triphosphate (m7GpppG)......Page 210
Synthesis of P1-guanosine-5' P3-(N2,N2,7-trimethyguanosine-5') triphosphate (m3227GpppG)......Page 212
Thermodynamic and apparent association constant......Page 215
Preparation of the samples......Page 217
Experimental conditions of fluorescence titration......Page 218
Corrections of fluorescence raw data......Page 219
Fluorescence data analysis......Page 221
Statistical analysis......Page 222
Activity of the nonenzymatic eIF4E protein......Page 223
Incorrectness of data linearization......Page 228
Principles of microcalorimetry experiments......Page 229
Calorimetric data treatment......Page 230
Calorimetric data analysis......Page 231
Principles of stopped-flow experiments......Page 232
Data analysis of ligand-binding kinetics......Page 233
Practical realization of fluorescence stopped-flow experiments......Page 236
Example of human eIF4E-m7GpppG association kinetics......Page 237
References......Page 239
Biophysical Studies of the Translation Initiation Pathway with Immobilized mRNA Analogs......Page 243
Generation of RNAs......Page 244
End labeling with biotinylated residues......Page 246
Chemical modification......Page 247
The use of immobilized RNAs in the BIAcore system......Page 248
The use of immobilized RNAs for atomic force microscopy......Page 251
Binding of the RNA construct to a gold-coated AFM slide......Page 253
Collecting AFM force spectroscopy data......Page 254
Analysis of AFM force spectroscopy data......Page 255
Protocol 1: 3'-End Biotinylation of a Transcript by Use of Poly (A) Polymerase......Page 256
Protocol 2: Introduction of Aldehyde Groups by Oxidation of the mRNA 3'-End......Page 257
Protocol 3: Thiolation at the mRNA 5'-End by Use of Polynucleotide Kinase......Page 258
References......Page 259
Protection-Based Assays to Measure Aminoacyl-tRNA Binding to Translation Initiation Factors......Page 261
Introduction......Page 262
Purification of Overproduced Initiator tRNA......Page 263
Protocols......Page 264
Notes......Page 265
Protocols......Page 266
Reagents......Page 267
Notes......Page 268
Protection Assay......Page 269
Protocols......Page 272
Data processing......Page 273
References......Page 275
NMR Methods for Studying Protein-Protein Interactions Involved in Translation Initiation......Page 278
Introduction......Page 279
Overview......Page 280
Types of interactions from the perspective of translation initiation......Page 283
Overview......Page 284
Protein size and NMR......Page 287
Slow exchange......Page 288
Equilibrium between free protein and complex......Page 290
Additional considerations and special cases......Page 292
Applications of NMR for studying protein interactions......Page 293
Testing for binding......Page 295
Controls......Page 296
False-negative results, or can NMR miss interactions?......Page 297
KD of the interaction......Page 298
Mapping of interaction surfaces......Page 300
Intermolecular NOEs......Page 301
Cross-saturation......Page 302
Paramagnetic labels......Page 303
Relaxation......Page 304
Methods for backbone assignments......Page 305
Methods for side-chain assignments......Page 308
Methods for structure determination......Page 309
Uniform isotope labeling......Page 311
Specific labeling schemes (nonuniform labeling)......Page 312
Segmental labeling......Page 313
Interdomain interactions and orientations......Page 314
Unstable proteins or complexes......Page 315
Summary......Page 316
References......Page 320
Structural Methods for Studying IRES Function......Page 327
Introduction......Page 328
A roadmap to answer specific questions......Page 329
Overview......Page 331
Materials......Page 333
Methods and procedures......Page 334
Quantitative......Page 335
Notes and hints......Page 337
5′ End labeling: Methods and procedures......Page 338
3′ End labeling: Methods and procedures......Page 339
RNase U2: Methods and procedures......Page 340
Overview......Page 341
Methods and procedures......Page 343
Overview......Page 344
Methods and procedures......Page 348
Notes and hints......Page 349
Overview......Page 350
Methods and procedures......Page 351
Notes and hints......Page 353
Overview......Page 354
Methods and procedures......Page 355
Overview......Page 358
Methods and procedures......Page 359
Notes and hints......Page 360
Conclusions......Page 361
References......Page 362
Biophysical and Biochemical Investigations of dsRNA-Activated Kinase PKR......Page 366
Introduction......Page 367
Expression and Purification of PKR......Page 369
RNA Synthesis......Page 372
Phosphorylation Assays......Page 373
Measuring RNA-Protein Stabilities......Page 376
Monitoring the Association State of PKR......Page 379
NMR Spectroscopy......Page 381
In Vitro Translation Assays......Page 385
Conclusions......Page 386
References......Page 387
Expression and Purification of Recombinant Wheat Translation Initiation Factors EIF1, EIF1A, EIF4A, EIF4B, EIF4F, EIF(iso)4F, and EIF5......Page 390
Introduction......Page 391
Cloning of cDNAs for wheat initiation factors......Page 392
Growth of E. coli and expression of initiation factors......Page 393
General protein purification procedures......Page 394
Purification of wheat eIF1......Page 396
Purification of wheat eIF4B......Page 397
Purification of eIF4G or eIF(iso)4G......Page 398
Results......Page 399
Conclusions......Page 400
References......Page 401
In Vitro Reconstitution and Biochemical Characterization of Translation Initiation by Internal Ribosomal Entry......Page 402
Introduction......Page 403
Chemicals, Enzymes, and Biological Materials......Page 404
Plasmids......Page 406
Buffers......Page 407
Preparation of IRES-Containing mRNA and Aminoacylated Initiator tRNA......Page 408
Strategies for Identification of Viral IRESs......Page 409
Buffers......Page 410
Buffers......Page 411
Identification of the Minimum Set of Factors Required for 48S Complex Formation......Page 414
Buffer......Page 416
Toe Printing to Map Stable Interactions of the IRES with Components of the Translation Apparatus......Page 417
Buffers......Page 420
Buffers......Page 425
References......Page 429