Contributors |
Preface |
Volumes in Series |
Transient Kinetics, Fluorescence, and FRET in Studies of Initiation of Translation in Bacteria / Pohl Milon ; Andrey L. Konevega ; Frank Peske ; Attilio Fabbretti ; Claudio O. Gualerzi ; Marina V. Rodnina1: |
Introduction |
Experimental Outline / 2: |
Materials / 3: |
Experimental Procedures / 4: |
Rapid Kinetic Measurements / 5: |
Quench-Flow Measurements / 6: |
GTPase Activity / 7: |
Dipeptide Formation / 8: |
Applications of the Method / 9: |
Binding of fMet-tRNA to the 30S Subunit / 10: |
FRET to IF3 / 11: |
Nucleotide Binding to IF2 / 12: |
Subunit Joining / 13: |
Acknowledgments |
References |
Binding of mRNA to the Bacterial Translation Initiation Complex / Sean M. Studer ; Simpson Joseph |
Real-Time Dynamics of Ribosome-Ligand Interaction by Time-Resolved Chemical Probing Methods / Anna Maria Giuliodori ; Cynthia L. Pon |
General Strategy |
Time-Resolved Chemical Probing with DMS |
Time-Resolved Probing with ONOOK |
Time-Resolved Chemical Probing with Fe(II)-EDTA |
Overexpression and Purification of Mammalian Mitochondrial Translational Initiation Factor 2 and Initiation Factor 3 / Domenick G. Grasso ; Brooke E. Christian ; Angela Spencer ; Linda L. Spremulli |
Assays for IF2[subscript MT] and IF3[subscript MT] |
Purification of His-Tagged IF2[subscript MT] |
Purification of His-Tagged IF3[subscript MT] |
In Vitro Studies of Archaeal Translational Initiation / Dario Benelli ; Paola Londei |
Preparation of S. solfataricus Cellular Extracts, Ribosomes, and Other Cellular Fractions |
In Vitro Translation of Archaeal mRNAs at High Temperature |
Ribosome/mRNA Interaction |
Initiation Factors |
Functional Analyses of Recombinant S. solfataricus IFS |
Interaction of Initiation Factors with Ribosomal Subunits |
IF/tRNA[subscript i superscript Met] Interaction |
Interaction of [[superscript 35]S]Met-tRNA[subscript i superscript Met] with Ribosomes |
Translational G-Proteins |
Reconstitution of Yeast Translation Initiation / Michael G. Acker ; Sarah E. Kolitz ; Sarah F. Mltchell ; Jagpreet S. Nanda ; Jon Lorsch |
Large-Scale Lysis of S. cerevisiae Cells |
Purification of 40S and 60S Ribosomal Subunits from S. cerevisiae |
Ribosome Quality Analysis (Identity Gel) |
Purification of His-Tagged eIF2 from S. cerevisiae |
Purification of His-Tagged eIF3 from S. cerevisiae |
Overexpression and Purification of Yeast eIF1, eIF1A, and eIF5 from E. coli |
Overexpression and Purification of Yeast eIF1A in E. coli |
Overexpression and Purification of Yeast eIF5B in E. coli |
Purification of Yeast Methionyl-tRNA Synthetase (YMETRS) from S. cerevisiae |
RNA Synthesis and Purification |
Charging tRNA[subscript iMet] with Methionine |
Filter Binding Assay to Monitor Ternary Complex Formation |
Benchtop eIF2 GTPase Assay / 14: |
43S/80S Complex Gel Shift Assay / 15: |
Solutions / 16: |
Assembly and Analysis of Eukaryotic Translation Initiation Complexes / Andrey V. Pisarev ; Anett Unbehaun ; Christopher U. T. Hellen ; Tatyana V. Pestova |
Chemicals, Enzymes, and Biological Materials |
Purification of 40S and 60S Ribosomal Subunits |
Purification of Native eIF3 and eIF4F |
Purification of Native eIF2, eIF5, and eIF5B |
Expression in E. coli and Purification of Recombinant eIF1, eIF1A, eIF4A, eIF4B, eIF4G, eIF5, and eIF5B |
Preparation and Aminoacylation of Initiator tRNA |
Assembly of 48S and 80S Initiation Complexes on [beta]-Globin mRNA |
Resolution of Translation Initiation Complexes by Sucrose Density Gradient Centrifugation |
Toe Printing Analysis of Ribosomal Complexes |
Site-Directed UV Cross-Linking of mRNA in Initiation Complexes |
Directed Hydroxyl Radical Probing to Localize Initiation Factors on the 40S Subunit |
Reconstitution of Mammalian 48S Ribosomal Translation Initiation Complex / Romit Majumdar ; Jayanta Chaudhuri ; Umadas Maitra |
Reagents |
Buffers |
General Methods |
Reconstitution of the 48S Ribosomal Complex |
Remarks |
Biophysical Approach to Studies of Cap-eIF4E Interaction by Synthetic Cap Analogs / Anna Niedzwiecka ; Janusz Stepinski ; Jan M. Antosiewicz ; Edward Darzynkiewicz ; Ryszard Stolarski |
Syntheses and Purification of Cap Analogs |
Binding of the Cap Analogs to eIF4E by Fluorescence Titration Experiments |
Application of Microcalorimetry to Studying eIF4E-Cap Interaction |
Stopped-Flow Fluorescence Studies of Binding Cap Analogs to eIF4E |
Biophysical Studies of the Translation Initiation Pathway with Immobilized mRNA Analogs / John E. G. McCarthy ; Steven Marsden ; Tobias von der Haar |
Generation of RNAs |
Protocol 1: 3[prime]-End Biotinylation of a Transcript by Use of Poly(A) Polymerase |
Protocol 2: Introduction of Aldehyde Groups by Oxidation of the mRNA 3[prime]-End |
Protocol 3: Thiolation at the mRNA 5[prime]-End by Use of Polynucleotide Kinase |
Protection-Based Assays to Measure Aminoacyl-tRNA Binding to Translation Initiation Factors / Yves Mechulam ; Laurent Guillon ; Laure Yatime ; Sylvain Blanquet ; Emmanuelle Schmitt |
Purification of Overproduced Initiator tRNA |
Preparation of Methionylated tRNA |
Protection Assay |
Conclusion |
NMR Methods for Studying Protein-Protein Interactions Involved in Translation Initiation / Assen Marintchev ; Dominique Frueh ; Gerhard Wagner |
Types of Interactions in Translation from the Perspective of K[subscript D] and Lifetime of the Complex |
NMR Methods for Studying Protein Interactions in Translation |
Descriptions of NMR Methods |
Special Cases |
Summary |
Structural Methods for Studying IRES Function / Jeffrey S. Kieft ; David A. Costantino ; Megan E. Filbin ; John Hammond ; Jennifer S. Pfingsten |
Methods |
Conclusions |
Biophysical and Biochemical Investigations of dsRNA-Activated Kinase PKR / Sean A. McKenna ; Darrin A. Lindhout ; Takashi Shimoike ; Joseph D. Puglisi |
Expression and Purification of PKR |
RNA Synthesis |
Phosphorylation Assays |
Measuring RNA-Protein Stabilities |
Monitoring the Association State of PKR |
NMR Spectroscopy |
In Vitro Translation Assays |
Expression and Purification of Recombinant Wheat Translation Initiation Factors eIF1, eIF1A, eIF4A, eIF4B, eIF4F, eIF(ISO)4F, and eIF5 / Laura K. Mayberry ; Michael D. Dennis ; M. Leah Allen ; Kelley Ruud Nitka ; Patricia A. Murphy ; Lara Campbell ; Karen S. Browning |
Procedures |
Results |
In Vitro Reconstitution and Biochemical Characterization of Translation Initiation by Internal Ribosomal Entry / Victoria G. Kolupaeva ; Sylvain de Breyne |
Plasmids |
Purification of Factors and Ribosomes |
Preparation of IRES-Containing mRNA and Aminoacylated Initiator tRNA |
Strategies for Identification of Viral IRESs |
Assays for 48S Complex Formation in Cell-Free Extracts: Sucrose Density Gradient Centrifugation |
Assays for 48S Complex Formation in Cell-Free Extracts: Toe Printing |
Identification of the Minimum Set of Factors Required for 48S Complex Formation |
Reconstitution of 48S Complex Formation on Type 2 Picornavirus IRESs |
Toe Printing to Map Stable Interactions of the IRES with Components of the Translation Apparatus |
Mapping Interactions of the IRES with Components of the Translation Apparatus by Chemical and Enzymatic Footprinting |
Directed Hydroxyl Radical Cleavage |
Author Index |
Subject Index |