Translation Initiation Extract Systems and Molecular Genetics

II......Page 1
V-IX......Page 2
XI-XIII......Page 7
XV......Page 10
XVII-XLII......Page 11
Use of Reticulocyte Lysates for Mechanistic Studies of Eukaryotic Translation Initiation......Page 37
Introduction......Page 38
mRNA......Page 39
Preparation of tRNA......Page 40
Translation of an mRNA to Yield a Radioactive Product......Page 41
Quantitation of Reaction Products......Page 42
Optimization of Translations......Page 44
Reporter Proteins for Translation......Page 46
Initiation mechanisms......Page 47
Competition between mRNAs......Page 48
Influence of variations of factor activity......Page 49
Sucrose gradients......Page 52
Toe printing......Page 54
References......Page 55
Introduction......Page 58
In Vivo Stabilization of Preinitiation Complexes by Formaldehyde Cross-Linking......Page 194
Preparation of Ovary Extracts......Page 60
Preparation of Embryo Extracts......Page 62
The Translation Assay......Page 63
Acknowledgments......Page 67
References......Page 212
Use of In Vitro Translation Extract Depleted in Specific Initiation Factors for the Investigation of Translational Regulation......Page 69
Factors Involved in Translation Initiation......Page 70
The Gal4-UAS system......Page 71
Preparation of fractionated lysates......Page 72
SDS-PAGE analysis......Page 73
Tertiary structure modeling......Page 74
In vitro translation in fractionated lysates......Page 75
Analysis of the cap dependency of translation of an mRNA......Page 76
Analysis of cap-dependent viral translational enhancer......Page 79
Analysis of cap-independent translation conferred by a viral 5'-leader......Page 82
References......Page 84
A Highly Efficient and Robust In Vitro Translation System for Expression of Picornavirus and Hepatitis C Virus RNA Genomes......Page 86
Introduction......Page 87
Buffers......Page 233
Sequence comparisons......Page 295
Experimental results......Page 89
Experimental Approaches to Determine Which Segments of an mRNA Are Shunted......Page 360
Media and solutions......Page 91
Other materials......Page 93
Methods for Cell-Free Synthesis of EMCV......Page 94
Determination of eIF4E protein levels......Page 154
Preparation of Krebs-2 S10 extract......Page 95
Analyzing translation......Page 96
EMCV RNA replication protocol......Page 97
Virus synthesis protocol......Page 98
HCV replication: An overview......Page 99
General characteristics of HCV RNA-directed translation and polyprotein processing in vitro......Page 100
Final Remarks......Page 101
HCV RNA in vitro translation protocol......Page 103
Characterizing NS3 protease inhibitors......Page 104
Probing glycosylation of HCV envelope proteins......Page 105
Protease protection assay for translocation of HCV envelope’proteins......Page 108
Perspectives and Future Applications......Page 109
References......Page 112
A Practical Approach to Isolate 48S Complexes: Affinity Purification and Analyses......Page 116
Introduction......Page 117
Design of Strepto-Tagged mRNAs for Affinity Purification of 48S Complexes......Page 119
RNP Immunoprecipitation (RIP) Assay......Page 274
Protocol 1......Page 121
Protocol 2......Page 122
Protocol 3......Page 124
Protocol 4......Page 127
Quantitative immunoblotting of 48S complexes......Page 128
Quantitative Northern blotting of 48S complexes......Page 131
Protocol 6: Analysis of eIF5-induced eIF release from purified 48S complexes......Page 132
Protocol 8: Sucrose density gradient analysis of 80S ribosome formation......Page 133
Acknowledgments......Page 135
References......Page 136
6......Page 138
Analysis of RNA:Protein Interactions In Vivo: Identification of RNA-Binding Partners of Nuclear Factor 90......Page 271
Introduction......Page 139
Quantitative Yeast Growth Assay......Page 142
Yeast strains......Page 143
Solutions......Page 144
Polysome profile analysis......Page 145
Procedures of quantitative growth assay (spot assay)......Page 146
Media......Page 147
Procedures......Page 148
Assay of Dominant Negative Mutants, Foreign Proteins, or Phenotypic Suppression by Overexpression......Page 149
Yeast expression plasmids......Page 150
Procedures......Page 152
Assay of Stringency in Start Codon Selection......Page 153
The role of RNA binding in function......Page 334
Cell growth and disruption......Page 155
Assay of Translation Initiation Activities with GCN4 as Reporter......Page 156
Yeast strains......Page 159
Gcn- phenotype test by 3AT sensitivity......Page 160
Use of drugs other than 3AT to study yeast Gcd- and Gcn- phenotypes......Page 161
Polysome Profiling......Page 162
Preparation of whole cell extracts......Page 164
Procedures of sucrose gradient-velocity sedimentation......Page 165
References......Page 166
7......Page 171
The Use of Fungal In Vitro Systems.for.Studying Translational Regulation......Page 232
Introduction......Page 172
The Use of Two-Hybrid Assay to Identify Protein-Protein Interaction Sites......Page 173
Position of the tethering site......Page 329
Procedure: transient transfection of mammalian cells with Photinus luciferase reporter constructs......Page 174
Preparation of N. crassa cell-free extracts for translation......Page 237
Western blot analysis......Page 175
Y2H......Page 177
GST Pull-Down Assay......Page 178
Plasmids......Page 179
Ribosome purification......Page 180
Preparation of the resin adsorbed to GST fusion proteins......Page 181
Binding reaction for binary interaction......Page 182
Determination of eIF4E mRNA levels......Page 185
Analysis of Fractionated Preinitiation Complexes......Page 186
Procedures of site-directed mutagenesis......Page 187
Yeast strains......Page 188
Cell culture and whole cell extract (WCE) preparation......Page 189
Anti-HA affinity resin preparation......Page 190
Acknowledgments......Page 191
Introduction......Page 195
Rationale Behind the Choice of HCHO as a Stabilization Agent......Page 197
Whole Cell Extract Preparation and WCE’Fractionation......Page 199
Non-P-element transposons......Page 201
Summary......Page 202
Heparin versus HCHO cross-linking......Page 203
Two percent HCHO cross-linking......Page 205
Trans-effects......Page 227
Polysome profile analysis and halfmers......Page 207
Molecular Genetic Structure-Function Analysis of Translation Initiation Factor eIF5B......Page 215
Introduction......Page 216
Site-directed and random mutagenesis of the FUN12 gene encoding yeast eIF5B......Page 218
Mutagenesis of tethered proteins can also be useful in identifying unique gain-of-function alleles......Page 341
Preparation of yeast cell extracts......Page 220
Yeast two-hybrid (Y2H) analysis......Page 221
Preparation of yeast cell extracts for in vitro translation assays......Page 222
Expression and purification of eIF5B from yeast......Page 223
Preparation of 5'-32P-labeled primer for toeprinting and sequencing reactions......Page 240
Purification of reassociated 80S ribosomes from yeast......Page 226
Strains used......Page 228
References......Page 230
Strains used......Page 236
Preparation of S. cerevisiae cell-free extracts for translation......Page 238
Method set 2......Page 241
Buffers......Page 243
Growth of culture and cell lysis......Page 244
Translation reactions......Page 245
Toeprint reaction protocol......Page 246
Analysis of translational regulation by upstream open reading frames......Page 247
Use of [35S]Met to examine polypeptide synthesis......Page 248
Analyses of premature translation termination by toeprinting samples of yeast translation reactions......Page 249
Effects of cycloheximide on ribosomal toeprint at a termination site......Page 250
Retroreinitiation after a premature termination requires functional Sup35p......Page 251
References......Page 252
Introduction......Page 255
P-Elements......Page 256
Imprecise P-element excision......Page 261
Use of upstream AUG codons as obstacles......Page 263
RNAi......Page 264
Important Sources for Drosophila Protocols......Page 266
References......Page 267
Introduction......Page 272
In Silico Detection and Analysis of eIF4E Family Members......Page 273
Procedure......Page 275
Results: G2/M phase cells......Page 276
Results: asynchronous cells......Page 279
Identification of Unknown RNAs by PCR Amplification and Sequencing......Page 280
Procedure......Page 281
Results......Page 282
Poly(A)-tailing procedure......Page 283
Results......Page 284
References......Page 286
Approaches for Analyzing the Differential Activities and Functions of eIF4E Family Members......Page 289
Introduction and Rationale......Page 290
Relationships among sequences......Page 297
Information derived from naturally occurring mutations......Page 298
Fluorescence quenching......Page 299
Affinity chromatography......Page 300
Expression of eIF4E Family Members......Page 301
Altering expression of eIF4E family members......Page 304
Function by complementation in yeast......Page 307
Recovery of translation in eIF4E-depleted mRNA-dependent translation systems......Page 309
Function of eIF4E family members in an mRNA-dependent reticulocyte translation system not depleted of eIF4E......Page 310
Likely candidates......Page 311
Source of "bait"......Page 314
Source of "prey"......Page 315
Far-Western analyses......Page 316
Mass spectrometry......Page 317
Global Microarray Studies of Polysomal mRNA Distribution......Page 318
References......Page 320
Tethered Function Assays: An Adaptable Approach to Study RNA Regulatory Proteins......Page 326
The MS2 bacteriophage coat protein as a tether......Page 330
N-peptide as a tether......Page 331
Use of hairpin structures as obstacles......Page 361
The Reporter mRNA......Page 332
The number and location of tethered binding sites......Page 333
Important Controls......Page 335
Separation of multiple functions that reside within the same protein......Page 339
Protein complexes: NMD......Page 340
Identifying mRNA localization functions and visualizing tagged mRNAs in vivo......Page 342
Analyzing mRNA modifying enzymes......Page 344
References......Page 345
Analysis of Ribosomal Shunting During Translation Initiation in Eukaryotic mRNAs......Page 349
Defining the Site or Sites of Ribosomal Recruitment......Page 350
Assessment of cap-dependent translation using hairpin structures......Page 352
Procedure: Ribonuclease protection assays......Page 354
Other approaches to block cap-dependent translation......Page 356
Procedure: cell-free translation......Page 357
Assessment of cap-independent translation......Page 358
Procedure: Assessing the integrity of an RNA hairpin structure......Page 362
Identification of Ribosomal Shunt Sites......Page 365
Isolation of 40S ribosomal subunits......Page 366
Nitrocellulose filter binding assays......Page 367
UV crosslinking and localization of crosslinked probes......Page 368
Assessing mRNA-rRNA Base Pairing in Yeast......Page 370
Evaluation of base pairing interactions between mRNA and 18S rRNA......Page 372
Assessing Ribosomal Shunting Mediated by mRNA-rRNA Base Pairing Interactions......Page 375
Considerations in Using the Mouse-Yeast Hybrid rRNA System......Page 377
References......Page 378
Author Index......Page 381
Subject Index......Page 396