Methods in Enzymology, Volume 467 : Part A: Volume 468 - Daniel Herschlag

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Methods in Enzymology, Volume 467

Part A: Volume 468

By: Daniel Herschlag

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Hardcover | 24 November 2009 | Edition Number 467

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496 Pages

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23.5 x 15.88 x 3.18

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This MIE volume provides laboratory techniques that aim to predict the structure of a protein which can have tremendous implications ranging from drug design, to cellular pathways and their dynamics, to viral entry into cells.

Expert researchers introduce the most advanced technologies and techniques in protein structure and folding
Includes techniques on tiling assays

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Non-Fiction Science Biology, Life Sciences Molecular Biology Biochemistry Enzymology Life Sciences in General Genetics excluding Medical

Contributors	p. xi
Preface	p. xv
Volumes in Series	p. xvii
Chemical and Functional Probing of RNA Structure, Interactions, and Folding	p. 1
Nucleotide Analog Interference Mapping	p. 3
Introduction	p. 4
Materials and Reagents	p. 7
Methods	p. 9
Properties of Analogs	p. 19
Nucleotide Analog Interference Suppression	p. 25
Conclusions	p. 26
References	p. 27
Hydroxyl-Radical Footprinting to Probe Equilibrium Changes in RNA Tertiary Structure	p. 31
Introduction	p. 32
Sample Preparation	p. 34
Equilibrium òOH Footprinting Based on Peroxidative Fenton Chemistry	p. 35
Equilibrium òOH Footprinting Based on Oxidative Fenton Chemistry	p. 36
Cleavage Product Separation	p. 38
Quantitation of the Changes in the Reactivity and Data Analysis	p. 40
Conclusions	p. 44
Acknowledgments	p. 44
References	p. 44
Rapid Quantification and Analysis of Kinetic òOH Radical Footprinting Data Using SAFA	p. 47
Introduction	p. 48
Using SAFA	p. 50
Data Normalization	p. 57
Data Visualization	p. 61
Conclusion	p. 64
Acknowledgment	p. 64
References	p. 65
High-Throughput SHAPE and Hydroxyl Radical Analysis of RNA Structure and Ribonucleoprotein Assembly	p. 67
Introduction	p. 68
Theory	p. 70
Practice	p. 73
Examples and Interpretation	p. 78
Perspectives and Conclusion	p. 86
Acknowledgments	p. 86
References	p. 87
Metal Ion-Based RNA Cleavage as a Structural Probe	p. 91
Introduction	p. 92
Mechanisms of Metal Ion-Based Cleavage of Nucleic Acids	p. 92
Metal Ion-Based Cleavage of RNA as a Structural Probe	p. 95
Protocols	p. 98
Acknowledgment	p. 103
References	p. 103
2'-Amino-Modified Ribonucleotides as Probes for Local Interactions Within RNA	p. 107
Introduction	p. 108
2'-Amino-2'-Deoxynucleotide Synthesis and Incorporation	p. 110
2'-Amino-2'-Deoxynucleotides as Sites for Covalent Modification	p. 111
General Strategy for Investigating 2'-Hydroxyl Interactions Using 2'-Deoxy and 2'-Aminonucleotides	p. 112
Studies of RNA Catalysis Using 2'-Amino-2'-Deoxynucleotides	p. 114
Using 2'-Aminonucleotides to Investigate RNA Structure and Function: Case Studies	p. 116
Conclusions	p. 121
Acknowledgments	p. 121
References	p. 122
RNA Crosslinking Methods	p. 127
Introduction	p. 128
Synthesis of Modified RNA Crosslinking Substrates	p. 129
Generation of Crosslinked RNAs	p. 135
Mapping of Crosslinked Nucleotides	p. 139
Assessing the Validity of Crosslinking Data	p. 141
References	p. 143
Chemical Probing of RNA and RNA/Protein Complexes	p. 147
Introduction	p. 148
Materials	p. 150
Handling of the Chemicals	p. 151
Optimization of the Chemical Probing Reactions	p. 152
Procedure of Chemical Probing	p. 154
RNA Extraction	p. 159
Normalization of the RNA Sample	p. 160
Primer Extension Analysis	p. 160
Data Evaluation	p. 162
Summary	p. 164
Acknowledgments	p. 164
References	p. 164
RNA Folding During Transcription: Protocols and Studies	p. 167
Introduction	p. 168
Protocol 1: Determination of Transcriptional Pause Sites	p. 169
Protocol 2: Structural Mapping of Paused Complexes	p. 172
Protocol 3: Cotranscriptional RNA Folding as Measured via Oligohybridization	p. 174
Protocol 4: Cotranscriptional RNA Folding Measured via P RNA Catalytic Activity	p. 175
Protocol 5: The Folding of Self-Cleaving RNAs During Transcription	p. 179
Additional Methodologies	p. 181
Cotranscriptional Folding Studies from our Laboratory	p. 181
References	p. 190
Catalytic Activity as a Probe of Native RNA Folding	p. 195
Introduction	p. 196
Preliminary Measurements of Catalytic Reaction	p. 199
Following RNA Folding by Continuous Activity Assay	p. 203
Following RNA Folding by Discontinuous Activity Assay	p. 206
Other Applications of Catalytic Activity as a Probe of Folding	p. 209
Acknowledgments	p. 215
References	p. 215
Probing RNA Structure Within Living Cells	p. 219
Introduction	p. 220
Experimental Procedure	p. 221
Application	p. 234
Limitations	p. 235
Conclusion	p. 236
Acknowledgments	p. 236
References	p. 236
Structural Analysis of RNA in Living Cells by In Vivo Synchrotron X-Ray Footprinting	p. 239
Introduction	p. 240
Beamline Setup for In Vivo Footprinting	p. 241
Preparation of Samples	p. 242
Exposure of Cells to X-Ray Beam	p. 244
Isolation of Total RNA from Irradiated Cells	p. 247
Primer Extension	p. 248
Analysis of X-Ray Footprinting Experiments	p. 251
Results on E. coli RNAs	p. 253
Future of Footprinting	p. 255
Acknowledgments	p. 255
References	p. 255
Determination of Intracellular RNA Folding Rates Using Self-Cleaving RNAs	p. 259
Introduction	p. 260
Using RNA Turnover Rates as a ôClockö for Measuring RNA Assembly Kinetics	p. 262
Applications	p. 280
Acknowledgments	p. 285
References	p. 285
Identifying Metal Ion Interactions in RNA	p. 287
Separation of RNA Phosphorothioate Oligonucleotides by HPLC	p. 289
Introduction: Phosphorothioate Oligonucleotides and the Need for Separation	p. 290
HPLC Separation of Phosphorothioate Diastereomers	p. 294
Materials and Methods	p. 298
Examples of Phosphorothioate Oligonucleotide Separations	p. 299
Acknowledgments	p. 307
References	p. 307
Use of Phosphorothioates to Identify Sites of Metal-Ion Binding in RNA	p. 311
Introduction	p. 312
Use of Phosphorothioate-Containing Ribozymes to Identify Sites of Metal-Ion Binding	p. 312
Protocols	p. 322
Acknowledgments	p. 330
References	p. 331
EPR Methods to Study Specific Metal-Ion Binding Sites in RNA	p. 335
Introduction	p. 336
Room Temperature EPR Spectroscopy to Quantify Mn²⁺ Bound to RNA	p. 341
Low-Temperature EPR Spectroscopy of Mn²⁺ Ions Bound to RNA	p. 345
ENDOR Spectroscopy to Identify Metal Ligands	p. 350
ESEEM Spectroscopy	p. 357
Summary	p. 361
Acknowledgments	p. 364
References	p. 364
RNA Thermodynamics	p. 369
Optical Melting Measurements of Nucleic Acid Thermodynamics	p. 371
Introduction	p. 371
Instrumentation	p. 372
Calibrations	p. 373
Brief Theory of Optical Melting Experiments	p. 375
Two-State Assumption	p. 378
C_p^°-Assumption	p. 378
Experimental Design	p. 378
Data Interpretation	p. 382
Error Analysis	p. 383
Summary	p. 384
Acknowledgments	p. 384
References	p. 384
Analyzing RNA and DNA Folding Using Temperature Gradient Gel Electrophoresis (TGGE) with Application to In Vitro Selections	p. 389
Introduction	p. 390
Temperature Gradient Gel Electrophoresis	p. 391
Experimental Design and Application of TGGE to RNA and DNA	p. 399
Acknowledgment	p. 406
References	p. 406
Studying RNA-DNA and RNA-Protein Interactions by Isothermal Titration Calorimetry	p. 409
Introduction	p. 410
Required Materials	p. 411
Instrumentation	p. 411
Sample Considerations and Preparation	p. 412
Cleaning the Sample Cell and Titration Syringe	p. 414
Collecting Titration Data	p. 415
Data Processing and Analysis	p. 418
Special Considerations	p. 420
Conclusions	p. 421
References	p. 422
Author Index	p. 423
Subject Index	p. 431
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Methods in Enzymology, Volume 467

Part A: Volume 468

At a Glance

Hardcover

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How to return your order

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You Can Find This Book In

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