Riboswitch Discovery, Structure and Function (eBook)

Donald H. Burke-Aguero (Herausgeber)

eBook Download: PDF | EPUB

2014 | 1. Auflage
570 Seiten
Elsevier Science (Verlag)
978-0-12-801335-9 (ISBN)

Infos

This new volume of Methods in Enzymology continues the legacy of this premier serial with quality chapters authored by leaders in the field. This volume covers research methods in riboswitch discovery and validation, synthesis and sample prep methods for large RNAs, riboswitch structure and function methods, folding pathways and dynamics, and ligand interactions and thermodynamics.

• Continues the legacy of this premier serial with quality chapters authored by leaders in the field
• Covers research methods in riboswitch discovery, structure and function
• Contains sections on such topics as riboswitch discovery and validation, synthesis and sample prep methods for large RNAs, riboswitch structure and function methods, folding pathways and dynamics, ligand interactions and thermodynamics

Inhalt

Front Cover 1
Riboswitch Discovery, Structure and Function 4
Copyright 5
Contents 6
Contributors 14
Preface 20
Volume 1 20
Volume 2 22
Part I: Riboswitch Discovery 26
Chapter One: Riboswitch Discovery by Combining RNA-Seq and Genome-Wide Identification of Transcriptional Start Sites 28
1. Introduction 29
2. RNA Isolation and mRNA Enrichment 32
2.1. Equipment and materials 32
2.2. RNA isolation and quality control 37
2.3. mRNA enrichment and quality control 38
3. Genome-Wide Mapping of Transcription Start Sites by dRNA-Seq 39
3.1. Equipment and materials 39
3.2. Hydrolysis of triphosphate groups at mRNA 5-ends by TAP 40
3.3. Ligation of adapter on 5-end of mRNAs 40
3.4. cDNA first strand synthesis by random priming 41
3.5. cDNA sizing on agarose gels 41
3.6. PCR amplification 42
3.7. Purification of the PCR products on Agencourt AMPure beads 43
3.8. Quality control of the libraries 44
3.9. Data analysis 44
4. Genome-Wide Analysis of Transcript Length by RNA-Seq 45
4.1. Strand-specific RNA-Seq library construction 45
4.2. Nonoriented whole-transcript RNA-Seq library preparation 46
5. Processing and Analysis of dRNA-Seq and RNA-Seq Data 47
5.1. Softwares and supplementary files required for the analysis 47
5.2. Protocol 48
5.2.1. Trimming of the adapter sequences 48
5.2.2. Creation of a reference index for bowtie 1 48
5.2.3. Alignment of the reads to the reference genome 49
5.2.4. Conversion of the SAM file to a sorted BAM file and creation of an index 49
5.2.5. Visualization of the alignments on a genome browser 49
6. Characterization of New Potential Riboswitches Using dRNA-Seq and RNA-Seq Analyses 49
References 51
Chapter Two: Discovering Human RNA Aptamers by Structure-Based Bioinformatics and Genome-Based In Vitro Selection 54
1. Introduction 54
2. Precautions 57
3. Generating a Human Genomic DNA Pool 57
3.1. Materials 57
3.1.1. High molecular weight human genomic DNA 57
3.1.2. Adapter oligonucleotide sequences 57
3.1.3. Enzymes 58
3.1.4. Buffers 59
3.1.4.1. Tris/borate/EDTA buffer (10x) 59
3.1.5. Instruments and miscellaneous 59
3.2. Procedures 59
3.2.1. Preparation of genomic DNA 59
3.2.2. Repairing genomic DNA ends 59
3.2.3. Addition of 5 phosphate group onto genomic DNA 60
3.2.4. Addition of 3 dA overhangs 60
3.2.5. Adapter ligation 60
3.2.6. PCR amplification 61
4. In Vitro Selection of RNA Aptamers 61
4.1. Materials 61
4.1.1. Selection buffers 61
4.1.2. Polyacrylamide gel electrophoresis 62
4.1.3. Agarose gel electrophoresis 62
4.1.4. Transcription 62
4.1.5. Reverse transcription 63
4.1.6. Polymerase chain reaction 63
4.1.7. Enzymes 63
4.1.8. Affinity column for in vitro selection 63
4.2. Procedure 63
4.2.1. Transcription 63
4.2.2. Purification of transcribed product 64
4.2.3. In vitro selection of RNA aptamers 66
4.2.4. Reverse transcription of selected RNAs 67
4.2.5. Polymerase chain reaction 67
5. Structure-Based Searches for Naturally Occurring Aptamers 69
5.1. Materials 69
5.1.1. Unix compliant operating system 69
5.1.2. RNABOB 69
5.1.3. RNArobo 69
5.2. Procedures 69
5.2.1. Descriptor 69
5.2.2. Sequence data 70
References 70
Part II: Synthesis and Sample Prep Methods for Large RNAs 72
Chapter Three: Affinity Purification of In Vitro Transcribed RNA with Homogeneous Ends Using a 3-ARiBo Tag 74
1. Introduction 75
2. Batch Affinity Purification of RNA Using a 3-ARiBo Tag 77
2.1. General scheme 77
2.2. Designing the ARiBo-fusion RNA 78
2.3. Cloning of the plasmid DNA template 79
2.4. Bacterial cell culture and plasmid preparation 80
2.5. In vitro transcription of RNA and optimization of glmS cleavage conditions 81
2.6. Batch affinity purification 82
2.7. Quantitative analyses for batch affinity purification using a 3-ARiBo tag 85
2.7.1. Denaturing gel electrophoresis 86
2.7.2. Quantitative analysis of ARiBo-fusion RNA produced by in vitro transcription 87
2.7.3. Quantitative analysis of glmS cleavage in the transcription reaction 87
2.7.4. Quantitative analysis of batch affinity purification 88
2.8. Troubleshooting 89
3. Ensuring 5-Homogeneity of Affinity-Purified RNA 92
3.1. General considerations in the selection of 5-sequences 94
3.2. Affinity purification of RNA using a 5-CRISPR tag and a 3-ARiBo tag 96
3.2.1. Bacterial expression of the Cse3 endonuclease 97
3.2.2. Purification of the Cse3 endonuclease 97
3.2.3. Cse3 endonuclease cleavage of the CRISPR-RNA-ARiBo precursor 100
3.3. Affinity purification of RNA using a 5-HH and a 3-ARiBo tag 101
3.4. Quantitative analyses when using a 5-tag 104
3.4.1. Quantitative analysis of 5-tag cleavage in the transcription reaction 104
3.4.2. Quantitative analysis of batch affinity purification 104
4. Summary 105
Acknowledgments 106
References 106
Chapter Four: Deoxyribozyme-Mediated Ligation for Incorporating EPR Spin Labels and Reporter Groups into RNA 110
1. Introduction 111
2. Synthesis of Spin-Labeled RNA Using Convertible Nucleosides 112
3. DNA-Catalyzed Ligation of RNA Using 9DB1* 115
3.1. General protocol for DNA-catalyzed RNA ligation on analytical scale for testing ligation sites and screening of liga... 117
3.1.1. Reagents 117
3.1.2. Procedure 118
4. Protocols for Synthesis of Spin-Labeled SAM-I Riboswitch 118
4.1. Synthesis of spin-labeled RNA (acceptor substrate) 119
4.1.1. Reagents 120
4.1.2. Procedure 120
4.2. In vitro transcription of donor substrate 121
4.2.1. Reagents 121
4.2.2. Procedure 122
4.3. Preparative DNA-catalyzed ligation of SAM-I RNA fragments 122
4.3.1. Reagents 123
4.3.2. Procedure 123
5. General Considerations and Future Developments 124
5.1. Choice of label position 124
5.2. Number and type of labels 124
5.3. Position and sequence context of ligation junction 125
5.4. Alternative DNA-catalyzed approaches for site-specific labeling of RNA 126
Acknowledgments 126
References 126
Chapter Five: A Flexible, Scalable Method for Preparation of Homogeneous Aminoacylated tRNAs 130
1. Introduction 130
2. Methods 132
2.1. tRNA aminoacylation using the flexizyme 132
2.2. Chemical protection of the aminoacyl bond 134
2.3. Purification of protected aminoacylated tRNA and deprotection 136
Acknowledgments 137
References 137
Chapter Six: Synthesis of a Biotinylated Photocleavable Nucleotide Monophosphate for the Preparation of Natively Folded RNAs 140
1. Theory 141
2. Equipment 143
3. Materials 143
3.1. Stock solutions and buffers 145
4. Protocol 146
4.1. Duration 146
4.2. Preparation 147
4.3. Caution 147
5. Step 1: Synthesis of Biotin-PC GMP 148
5.1. Overview 148
5.2. Duration 148
5.2.1. Synthesis of PC alkyne 148
5.2.1.1. Tip 149
5.2.1.2. Tip 149
5.2.1.3. Tip 149
5.2.2. Synthesis of PC alkyne GMP 149
5.2.2.1. Tip 150
5.2.3. Synthesis of biotin-PC GMP 150
5.2.3.1. Tip 151
5.2.3.2. Tip 151
6. Step 2: Transcription of D5 and Ribosomal A-site RNAs Using Unmodified GTP and Biotin-PC GMP 151
6.1. Overview 151
6.2. Duration and transcription optimization 151
6.3. Tip 153
6.4. Tip 153
7. Step 3: Purification of Biotin-Labeled RNA with Affinity Avidin Column and Photocleavage 153
7.1. Overview 153
7.2. Duration 154
7.3. Tip 154
7.4. Tip 154
8. Conclusions 154
Acknowledgments 156
References 156
Chapter Seven: Chemo-Enzymatic Synthesis of Selectively 13C/15N-Labeled RNA for NMR Structural and Dynamics Studies 158
1. Theory 160
2. Equipment 163
3. Materials 164
3.1. Solutions and buffers 166
4. Protocol 169
5. Step 1: Synthesis of Uracil 170
5.1. Overview 170
5.2. Duration 170
5.3. Tip 172
5.4. Tip 172
5.5. Tip 172
5.6. Tip 172
6. Step 2: Synthesis of UTP 173
6.1. Overview 173
6.2. Duration 173
6.3. Tip 174
6.4. Tip 174
6.5. Tip 174
6.6. Tip 174
6.7. Tip 175
7. Step 3: Synthesis of CTP 175
7.1. Overview 175
7.2. Duration 175
7.3. Tip 176
7.4. Tip 176
7.5. Tip 176
8. Step 4: Purification and Quantification 176
8.1. Overview 176
8.2. Duration 176
8.3. Tip 178
8.4. Tip 178
8.5. Tip 178
8.6. Tip 178
8.7. Tip 178
8.8. Tip 178
9. Step 5: Quality Control 179
9.1. Overview 179
9.2. Duration 179
9.3. Tip 180
9.4. Tip 180
10. Step 6: In Vitro RNA Transcription 180
10.1. Overview 180
10.2. Duration 181
10.3. Tip 182
10.4. Tip 182
11. Step 7: NMR Applications 182
11.1. Overview 182
11.2. Heteronuclear single quantum coherence (HSQC) 183
11.3. Transverse relaxation optimized spectroscopy (TROSY) 183
12. Conclusion 183
Acknowledgments 186
References 186
Part III: Structure and Folding 188
Chapter Eight: SHAPE Analysis of Small RNAs and Riboswitches 190
1. Theory 191
2. Equipment 192
3. Materials 192
3.1. Solutions and buffers 193
4. Protocol 194
4.1. Preparation 194
4.2. Duration 195
5. Step 1: RNA Folding and SHAPE Probing 195
5.1. Overview 195
5.2. Duration 196
5.3. Tip 197
5.4. Tip 197
5.5. Tip 197
6. Step 2: Primer Extension 197
6.1. Overview 197
6.2. Duration 197
6.3. Tip 198
6.4. Tip 198
6.5. Tip 199
6.6. Tip 199
7. Step 3: Capillary Electrophoresis 199
7.1. Overview 199
7.2. Duration 199
7.3. Tip 199
7.4. Tip 200
7.5. Tip 200
8. Step 4: Data Processing Using QuShape 200
8.1. Overview 200
8.2. Duration 200
8.3. Tip 205
8.4. Tip 205
8.5. Tip 206
8.6. Tip 206
8.7. Tip 206
8.8. Tip 206
8.9. Tip 207
9. Step 5: Data Processing and RNA Modeling 207
9.1. Overview 207
9.2. Duration 207
9.3. Tip 209
9.4. Tip 211
9.5. Tip 211
Acknowledgments 211
References 211
Chapter Nine: Experimental Approaches for Measuring pKas in RNA and DNA 214
1. Introduction 215
2. Experimental Parameters for pH Titrations 218
2.1. Potential pitfalls: pH-promoted RNA unfolding, RNA degradation, and poor baselines 218
2.2. Choosing the pH probe and meter 221
2.3. Whether to use a buffer 222
2.4. Corrections to the pH meter reading and the use of pH paper 223
2.5. Choosing an experimental method and assigning the pKa 223
3. RNA Cleavage Kinetics 224
3.1. Ribozyme cleavage 224
3.2. Chimeric oligonucleotide cleavage 228
4. Spectroscopic-Detected Methods 229
4.1. General considerations for spectroscopic-detected pH titrations 229
4.2. UV absorbance-detected pH titrations 230
4.3. Fluorescence-detected pH titrations 232
4.4. NMR-detected pH titrations 235
4.5. Raman crystallography pH titrations 239
5. Perspective 240
Acknowledgments 241
References 241
Chapter Ten: Crystallographic Analysis of TPP Riboswitch Binding by Small-Molecule Ligands Discovered Through Fragment-Ba... 246
1. Introduction 247
2. Methods 248
2.1. Growth of riboswitch-fragment co-crystals 248
2.1.1. Considerations in transcription template and RNA construct design 249
2.1.2. Considerations in fragment selection 249
2.1.3. In vitro transcription of TPP riboswitch RNA 251
2.1.4. Initial screens of crystallization conditions with the cognate ligand 251
2.1.5. Initial screens for fragment co-crystals 252
2.1.6. Development of cryoprotectant solutions for vitrification of fragment co-crystals 252
2.2. Structure solution by molecular replacement 254
2.2.1. Structure solution by molecular replacement 255
2.2.2. Model building and refinement 255
2.2.3. Building the fragment into the model 255
3. Conclusions 256
Acknowledgments 256
References 256
Chapter Eleven: Methods for Using New Conceptual Tools and Parameters to Assess RNA Structure by Small-Angle X-Ray Scattering 260
1. Introduction 261
2. Specialized Equipment 263
3. Preparation of the RNA for a SAXS Study 263
3.1. Assessing the folded state of the RNA 263
3.2. Importance of buffer subtraction 269
4. Interpretation of the X-Ray Scattering Curve 270
4.1. Quantitating compactness 271
4.2. SAXS invariants 274
4.3. Real-space parameters 274
4.4. Dimensionless Kratky plot 275
5. Case Studies 277
5.1. SAM-I riboswitch 278
5.2. LYS riboswitch 279
6. Multiphase Volumetric Modeling 280
6.1. B12 riboswitch 281
7. Gold Labels and Comprehensive Conformations 281
8. Considerations 283
Acknowledgments 285
References 285
Part IV: Dynamics 290
Chapter Twelve: Use of 19F NMR Methods to Probe Conformational Heterogeneity and Dynamics of Exchange in Functional RNA M ... 292
1. Introduction 293
2. Methods 295
2.1. Sample design 295
2.2. Sample preparation 296
2.3. One-dimensional 19F experiments to identify the distribution of folds 296
2.4. Two-dimensional 19F-19F EXSY experiments to measure conformational exchange 297
2.5. Application to analysis of distribution and exchange in a bistable RNA stem loop 300
2.6. Application to analysis of distribution and exchange in a biologically significant system 301
3. Conclusion and Remarks 307
Acknowledgments 308
References 308
Chapter Thirteen: Site-Directed Spin-Labeling Strategies and Electron Paramagnetic Resonance Spectroscopy for Large Ribos ... 312
1. Techniques Used for Riboswitch Studies 315
1.1. Biochemical 315
1.2. Spectroscopy and labeling 317
1.2.1. EPR 317
1.3. Site-directed spin labeling 318
1.3.1. Labeling positions 318
1.3.2. Choice of spin label 320
1.3.3. SDSL for CW and pulsed EPR 321
1.3.4. Advantages/disadvantages 321
2. Ligation Methods for SDSL of Large Riboswitches 322
2.1. T4 DNA ligase 323
2.2. Considerations for SDSL and T4 DNA-mediated ligation of large riboswitches 324
2.2.1. Optimizing conditions 325
2.2.2. Protocol 326
2.2.2.1. Synthetic RNA preparations 326
2.2.2.1.1. Deprotection of synthetic RNA 327
2.2.2.1.2. Spin labeling of synthetic RNA 327
2.2.2.2. Transcribed RNA preparations 328
2.2.2.2.1. Dephosphorylation and monophosphorylation 328
2.2.2.3. Small- and large-scale ligations 328
2.2.2.3.1. Annealing 329
2.2.2.3.2. Ligation 329
2.2.2.3.3. Scaling up ligation reactions 330
2.2.2.4. Large-scale purification of ligation product 331
2.2.2.4.1. Large-scale PCA extraction 331
2.2.2.4.2. Large-scale ethanol precipitation 331
2.2.2.4.3. Purification by dPAGE 331
2.2.2.4.4. Sample preparation for CW-EPR 332
3. CW-EPR Spectral Analysis of Riboswitches 332
References 334
Chapter Fourteen: Using sm-FRET and Denaturants to Reveal Folding Landscapes 338
1. Introduction 339
2. Single-Molecule FRET: Technical Aspects 342
3. Riboswitch Structure and Biological Function 344
3.1. sm-FRET studies of the adenine aptamer under nondenaturing conditions 346
4. Combination of sm-FRET and Denaturants to Investigate Riboswitch Folding 348
4.1. Urea-induced perturbation of RNA folding: Ensemble studies 349
4.2. Urea-induced perturbation of RNA folding: Single-molecule studies 350
4.3. Technical considerations when combining sm-FRET and chemical denaturants 351
4.4. Urea-induced effects on the single-molecule dynamics of adenine aptamers 353
4.5. In situ cycling between Mg2+ and urea: A method to quantify the reversibility of chemical denaturation 354
4.6. Methods for comparing Mg2+-assisted folding and urea-induced unfolding 354
4.7. Influence of urea on the undocking rates: A method to quantify the ligand-induced stabilization of the aptamer domain 355
4.8. Influence of urea on the docking rates: A method to evaluate the rate-limiting step for folding 358
5. Summary and Prospects 362
References 362
Chapter Fifteen: Riboswitch Structure and Dynamics by smFRET Microscopy 368
1. Introduction 369
1.1. Single-molecule fluorescence resonance energy transfer 372
1.1.1. Advantages of single-molecule methods 372
1.1.2. Fluorescence resonance energy transfer 374
2. Methods 376
2.1. Labeling and purification of riboswitches 376
2.2. Preparation of quartz slides 379
2.3. Surface attachment and oxygen scavenging systems 381
2.4. smFRET using prism-based TIRF microscopy 382
2.5. Heat-annealing of riboswitch RNAs 383
3. Practical Experimental Considerations 384
4. Data Analysis 385
4.1. FRET histograms 386
4.2. Kinetic analysis 388
4.3. Cross-correlation analysis 390
5. Induced-Fit Versus Conformational Selection 390
6. Summary and Conclusions 393
Acknowledgments 393
References 393
Chapter Sixteen: Ribosome Structure and Dynamics by smFRET Microscopy 400
1. Introduction 401
2. Overview of Ribosome Structure and Function 402
3. Methodology 404
3.1. What is out there? 404
3.2. Why single-molecule approaches? 405
3.3. Why is the ribosome an ideal system for smFRET? 406
4. Ribosome Dynamics 409
4.1. Choosing a question 410
4.2. Choosing a dye 410
4.3. Using phylogenetic analysis and structural modeling to guide choice of labeling sites 411
4.4. Fluorescently labeling various translation components 414
4.4.1. tRNA labeling 414
4.4.2. Ribosome labeling 414
4.4.3. Translation factors labeling 416
4.5. Testing activity of purified translation components 416
4.5.1. Filter binding and puromycin reactivity assay 416
4.6. Assessing the spectroscopic properties of the labeled components 417
4.7. Ribosomal complex assembly 418
4.8. Immobilization schemes 418
4.9. Imaging 419
5. Data Acquisition 420
5.1. Selecting a camera 420
5.2. Signal to noise 420
5.2.1. Dark noise, readout noise, Poisson noise 420
5.2.2. Exposure time, QE, DR, gain 421
5.2.3. Binning 421
5.2.4. Nyquist theorem: Undersampling, oversampling 422
5.2.5. Bit depth 423
5.2.6. Improving temporal resolution 423
5.3. Acquisition 423
5.3.1. Analysis algorithms 424
6. Building and Verifying Histograms, Normalization, Gaussian Fitting 425
7. Future Directions 425
References 426
Chapter Seventeen: Unraveling the Thermodynamics and Kinetics of RNA Assembly: Surface Plasmon Resonance, Isothermal Titr... 432
1. The RNA Folding Problem and Assembly of RNA Tertiary Structure 433
2. Practical Aspects of Biophysical Studies of RNA Assembly 435
2.1. RNA assembly: Specification and control of ionic conditions 435
2.1.1. Protocol 1: Large-scale RNA purification and buffer exchange via dialysis 437
2.2. RNA assembly: Binding parameters and choice of methodology 438
2.3. RNA assembly: Analytical aspects and experimental design 439
3. Specific Measurements of Ion-Driven RNA Assembly 442
3.1. Ion dependence of assembly: CD 442
3.1.1. Protocol 2 444
3.2. Rates of intermolecular assembly: SPR 446
3.2.1. Protocol 3 447
3.3. Thermodynamics of RNA assembly: ITC 449
3.3.1. ITC experimental design 451
3.3.2. Experimental design: Buffer match 453
4. Concluding Remarks 454
Acknowledgments 454
References 454
Part V: Ligand Interactions 458
Chapter Eighteen: ITC Analysis of Ligand Binding to PreQ1 Riboswitches 460
1. Introduction 461
1.1. Information content in an ITC experiment 463
2. Experimental Procedures for ITC 464
2.1. Assessing the feasibility of ITC experiments 464
2.2. Instrumentation, materials, and solutions for ITC 466
2.3. RNA and ligand preparation 467
2.4. The isothermal titration calorimetry experiment 468
2.5. ITC data analysis 469
2.6. Manual adjustment of the baseline 471
2.7. Publishing ITC results and representative analysis 471
Acknowledgments 473
References 474
Chapter Nineteen: Facile Characterization of Aptamer Kinetic and Equilibrium Binding Properties Using Surface Plasmon Res... 476
1. Introduction 477
2. Materials 478
2.1. Instrumentation 478
2.2. Sensor surface immobilization 478
2.3. Aptamer binding assay 479
3. Sensor Surface Immobilization 480
3.1. Pre-concentration assay 480
3.2. DNA linker immobilization 482
4. Characterization of Aptamer Binding Properties 483
4.1. Aptamer design and preparation 483
4.2. Startup cycles 484
4.3. Aptamer binding assay 486
4.4. Analysis of aptamer binding properties 488
5. Conclusion 490
Acknowledgments 491
References 491
Chapter Twenty: The AdoCbl-Riboswitch Interaction Investigated by In-Line Probing and Surface Plasmon Resonance Spectrosc... 492
1. Introduction 493
2. In-Line Probing Experiments 496
2.1. Mechanism of the in-line probing reaction 496
2.2. Performing an in-line probing experiment 497
2.2.1. Overview and general remarks 497
2.2.2. Equipment for in-line probing 497
2.2.3. Buffers and solutions for in-line probing 498
2.2.4. In-line probing reaction and PAGE analysis 498
2.2.5. Data analysis and calculation of KD values 499
3. SPR Spectroscopy 502
3.1. The method of SPR 502
3.2. Practical example: Studying the AdoCbl-btuB riboswitch interaction by SPR 503
3.2.1. Overview and general remarks 503
3.2.2. Equipments for SPR 504
3.2.3. Buffers and solutions for SPR 504
3.2.4. Sample preparation 505
3.2.5. Immobilization of the RNA on sensor surface 505
3.2.6. SPR measurements 507
3.2.7. Data analysis 509
4. Conclusion 509
Acknowledgments 510
References 510
Chapter Twenty-One: Assessing RNA Interactions with Proteins by DRaCALA 514
1. Introduction 515
2. DRaCALA-Based Detection of Protein-Ligand Interactions 516
3. Principle of DRaCALA 518
4. Determination of Fraction Bound by DRaCALA 518
5. Steps for Performing DRaCALA to Detect Protein Interaction With RNA 520
5.1. Procedure: Preparation of expression vector 520
5.1.1. Reagents 520
5.1.2. Method 520
5.2. Procedure: Preparation of whole cell lysates 521
5.2.1. Reagents 521
5.2.2. Method 521
5.3. Procedure: Template generation 522
5.3.1. Reagents 522
5.3.2. Method 522
5.3.3. Consideration for template generation 523
5.4. Procedure: In vitro transcription of RNA 523
5.4.1. Reagents 523
5.4.2. Method 524
5.5. Procedure: 5-end labeling of RNA 525
5.5.1. Reagents 525
5.5.2. Method: AnP treatment to remove 5-triphosphate 526
5.5.3. Method: 5-end labeling of RsmY and RsmZ 527
5.5.4. Consideration for labeling of RNA ligand 528
5.6. Procedure: Determining protein-RNA interaction 528
5.6.1. Reagents 528
5.6.2. Method: Binding reaction and spotting of DRaCALA spots 529
5.7. Procedure: Determining relative affinity 532
5.7.1. Method 532
5.8. Procedure: Determining specificity of binding through competition 533
5.8.1. Method 533
6. CsrA Binds Specifically to RsmY and RsmZ 534
7. Other Modifications of DRaCALA for RNA-Protein Interactions 535
References 536
Author Index 538
Subject Index 560
Color Plate 572

Textauszug

Preface

Donald H. Burke-Aguero

The early years of the twenty-first century have seen an explosion of interest in the diverse capabilities of RNA. Riboswitches capture the excitement and promise of this field. They are structurally dynamic, they sense and respond to specific molecular partners, their occupancy states governs gene regulatory decisions, and they can be engineered to reprogram gene regulatory circuitry. Importantly, many of the experimental and theoretical tools that have been used to study riboswitches can also be applied to other RNAs, and tools developed for studies of other RNAs can be applied to riboswitches.

These two volumes (Methods in Enzymology 549 and 550) include 40 contributions that outline cutting-edge methods representing a wide spectrum of research questions and scientific themes. The first volume emphasizes natural riboswitches, from their discovery to assessment of their structures and functions. The second volume shifts the focus to applying riboswitches as tools for a variety of applications and as targets for inhibition by potential new antibacterial compounds. A third volume (Methods in Enzymology 553) will appear shortly after these two focusing on computational methods for predicting and evaluating dynamic RNA structures. Although the chapters are organized into discrete themes, many cut across thematic boundaries by weaving together diverse methodological solutions and several of the chapters could fit comfortably into more than one section.

Volume 1

Riboswitch discovery. In the early days of the riboswitch field, new riboswitches were discovered at a frenetic pace, often by comparing large sets of bacterial genomes. While that approach continues to identify new members of known riboswitch families, the pace has slowed, and new discovery methods are needed. The series begins with two chapters outlining new methods that utilize informatics approaches in combination either with RNASeq and genome-wide methods (Rosinski-Chupin) or with in vitro selection (Ho) to discover new natural riboswitches.

Sample preparation. Any effort to characterize purified, functional RNAs will only be as good as the corresponding sample preparations. Therefore, the next five chapters are dedicated to methods for the synthesis and preparation of large RNAs. Three groups exploit specialty nucleic acids with functionalities of their own. The first chapter in this set describes the use of cotranscribed aptamer affinity tags that are removed by activatable self-cleavage (Di Tomasso). This is followed by methods for using catalytic deoxyribozyme ligases to assemble large RNAs from synthetic fragments, some of which carry site-specific spin labels for electron paired resonance studies (Wawrzyniak-Turek). The third chapter in this set describes the combined use of aminoacyl transferase ribozymes and chemical protection to generate charged tRNAs on a large scale (Zhang). These are followed by two chapters that integrate organic chemical methods with improved enzymology to produce photocleavable biotinylated guanosine that incorporates at the 5′ end of in vitro transcripts (Luo) and large quantities of selectively 13C/15N-labeled RNA in previously unattainable labeling patterns for improved spectroscopic analysis (Alvarado).

Structure and function. The biochemical functions of riboswitches are inextricably linked with their three-dimensional structures. The next several chapters, therefore, provide methods for evaluating riboswitch structure and function. Updated protocols are provided for the widely utilized SHAPE method of structural probing, along with details of how to implement new software for data interpretation (Rice). It is well recognized that structural context can perturb pKa values within RNA and DNA; hence, the next chapter details how to measure them without falling into traps of oversimplifying the underlying molecular processes (Taplyal). The next chapter provides methods for obtaining appropriate crystals for ligand–RNA complexes, with emphasis on fragment-bound TPP riboswitches (Warner). This section ends with a detailed description of experimental and analytical methods for using small-angle X-ray scattering to define RNA conformations in solution (Reyes).

Conformational dynamics. Spectroscopic methods are ideal for following riboswitch conformational dynamics in real time. The first two chapters of this section describe site-specific incorporation of spectroscopic labels and their use in addressing specific question, first with 19F NMR to probe conformational exchange (Zhao) and then with spin-label probes for electron paramagnetic resonance spectroscopy of large RNAs (Esquiaqui). Single-molecule methods such as smFRET have become a staple of modern biophysical analysis. Three chapters provide detailed guidance on many facets of smFRET, from sample preparation, data acquisition, and analysis to explorations of folding landscapes (Shaw, Suddala, and Shebl). The last chapter of this section describes how to integrate surface plasmon resonance (SPR), isothermal titration calorimetry, and circular dichroism to examine tertiary docking (Hoogstraten).

Ligand interactions. One of the most important characteristics of riboswitches is their ability to sense the presence of specific metabolites by forming bound molecular complexes. Isothermal titration calorimetry is one of the most powerful methods for evaluating the energetics of RNA–small molecule complexes (Wedekind). SPR is another powerful tool for characterizing aptamer kinetic and equilibrium binding properties and is detailed in two chapters (Chang and Schaffer). Finally, an innovative and relatively new technique known as DRaCALA is described in the last chapter of the first volume (Patel).

Volume 2

The second volume in this series takes a different perspective on riboswitches. Specifically, now that nature has shown us that RNA modules can sense metabolites and report on them, how can we take advantage of that ability to engineer new properties into cells and biochemical systems? Necessarily, this volume takes a much broader view of riboswitches than those found in nature, encompassing ligand-responsive transcriptional and translational modules, ribozymes, sensors, and modules that induce fluorescence in a fluorophore upon formation of the bound complex. It encompasses Synthetic Biology applications as tools to understand normal biological processes, and as tools to reprogram metabolite flux in workhorse organisms. Finally, it comes full circle by screening small-molecule libraries for inhibitors of natural riboswitches.

In short, this second volume details methods at the cutting edge of the translational science of riboswitches.

Artificial riboswitches. The first six chapters of the second volume provide methods for several approaches to construct and optimize artificial riboswitches. There has been substantial progress toward designing artificial riboswitches from scratch, especially when guided by experimental validation (Moerl). A contrasting approach uses in vitro selection/evolution to obtain ligand-responsive ligase ribozymes from highly diverse starting populations (Olea), or to reshape and reprogram the ligand-binding and expression platforms of natural riboswitches (Batey). The next chapter presents methods for optimizing signal transduction, since regulation sometimes benefits from maximizing suppression of basal expression in the OFF state and sometimes from maximizing expression in the ON state (Goodson). The next two chapters address optimization in two very different cell-free systems, first using coupled transcription–translation to optimize a ligand-responsive self-cleaving ribozyme, or “aptazyme” (Ichihashi), and then taking advantage of a eukaryotic mechanism by which ribosomes “shunt” past certain secondary structures, which can be stabilized to increase shunting efficiency by binding to the analyte ligand (Ogawa).

Ligand-responsive fluorescent sensors. There has been longstanding interest in coupling the binding of ligands to RNA with the emission of light. One such system is that of the recently described Spinach (and Spinach2) aptamer mimics of green fluorescent protein, which are the focus of the next five chapters, each in a different system. The first chapter in this section, from the lab that discovered and first described the Spinach system, presents methods for using it to image intracellular RNA in mammalian cells (Strack). The next two chapters describe how to use these modules in bacterial cells, first as intracellular sensors of intracellular cyclic dinucleotide levels (Kellenberger) and then for simultaneous and independent monitoring of mRNA and protein levels (Pothoulakis). The next chapter takes this same question into solution and into vesicle-based artificial cells (van Nies). The fifth chapter in this section couples sensing of oligonucleotide “ligands” with Spinach2 output in real time for sequence-specific target quantitation and potential point-of-care applications (Bhadra).

Synthetic biology: Conditional control of gene expression. The third section of this volume lays out several methods for using artificial or natural riboswitches to study gene function. This has proven to be a powerful tool in organisms for which limited genetic tools are available, such as the intracellular pathogen Mycobacteria (Van Vlack), as well as in more readily manipulated, nonpathogenic bacteria such as Streptomyces coelicolor (Rudolph). Eukaryotes...

Produkt-Details

• Erscheint lt. Verlag: 21.11.2014
• Sprache: englisch
• Themenwelt: Medizin / Pharmazie ► Medizinische Fachgebiete
• Themenwelt: Naturwissenschaften ► Biologie ► Biochemie
• Themenwelt: Naturwissenschaften ► Biologie ► Genetik / Molekularbiologie
• Themenwelt: Naturwissenschaften ► Physik / Astronomie ► Angewandte Physik
• ISBN-10: 0-12-801335-4 / 0128013354
• ISBN-13: 978-0-12-801335-9 / 9780128013359