Save up to 30% on Elsevier print and eBooks with free shipping. No promo code needed.
Save up to 30% on print and eBooks.
Riboswitch Discovery, Structure and Function
1st Edition, Volume 549 - November 21, 2014
Editor: Donald H. Burke-Aguero
Language: English
Hardback ISBN:9780128011225
9 7 8 - 0 - 1 2 - 8 0 1 1 2 2 - 5
eBook ISBN:9780128013359
9 7 8 - 0 - 1 2 - 8 0 1 3 3 5 - 9
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 method…Read more
Purchase options
LIMITED OFFER
Save 50% on book bundles
Immediately download your ebook while waiting for your print delivery. No promo code is needed.
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
Biochemists, biophysicists, molecular biologists, analytical chemists, and physiologists.
Preface
Volume 1
Volume 2
Part I: Riboswitch Discovery
Chapter One: Riboswitch Discovery by Combining RNA-Seq and Genome-Wide Identification of Transcriptional Start Sites
Abstract
1 Introduction
2 RNA Isolation and mRNA Enrichment
3 Genome-Wide Mapping of Transcription Start Sites by dRNA-Seq
4 Genome-Wide Analysis of Transcript Length by RNA-Seq
5 Processing and Analysis of dRNA-Seq and RNA-Seq Data
6 Characterization of New Potential Riboswitches Using dRNA-Seq and RNA-Seq Analyses
Chapter Two: Discovering Human RNA Aptamers by Structure-Based Bioinformatics and Genome-Based In Vitro Selection
Abstract
1 Introduction
2 Precautions
3 Generating a Human Genomic DNA Pool
4 In Vitro Selection of RNA Aptamers
5 Structure-Based Searches for Naturally Occurring Aptamers
Part II: Synthesis and Sample Prep Methods for Large RNAs
Chapter Three: Affinity Purification of In Vitro Transcribed RNA with Homogeneous Ends Using a 3′-ARiBo Tag
Abstract
1 Introduction
2 Batch Affinity Purification of RNA Using a 3′-ARiBo Tag
3 Ensuring 5′-Homogeneity of Affinity-Purified RNA
4 Summary
Acknowledgments
Chapter Four: Deoxyribozyme-Mediated Ligation for Incorporating EPR Spin Labels and Reporter Groups into RNA
Abstract
1 Introduction
2 Synthesis of Spin-Labeled RNA Using Convertible Nucleosides
3 DNA-Catalyzed Ligation of RNA Using 9DB1*
4 Protocols for Synthesis of Spin-Labeled SAM-I Riboswitch
5 General Considerations and Future Developments
Acknowledgments
Chapter Five: A Flexible, Scalable Method for Preparation of Homogeneous Aminoacylated tRNAs
Abstract
1 Introduction
2 Methods
Acknowledgments
Chapter Six: Synthesis of a Biotinylated Photocleavable Nucleotide Monophosphate for the Preparation of Natively Folded RNAs
Abstract
1 Theory
2 Equipment
3 Materials
4 Protocol
5 Step 1: Synthesis of Biotin-PC GMP
6 Step 2: Transcription of D5 and Ribosomal A-site RNAs Using Unmodified GTP and Biotin-PC GMP
7 Step 3: Purification of Biotin-Labeled RNA with Affinity Avidin Column and Photocleavage
8 Conclusions
Acknowledgments
Chapter Seven: Chemo-Enzymatic Synthesis of Selectively 13C/15N-Labeled RNA for NMR Structural and Dynamics Studies
Abstract
1 Theory
2 Equipment
3 Materials
4 Protocol
5 Step 1: Synthesis of Uracil
6 Step 2: Synthesis of UTP
7 Step 3: Synthesis of CTP
8 Step 4: Purification and Quantification
9 Step 5: Quality Control
10 Step 6: In Vitro RNA Transcription
11 Step 7: NMR Applications
12 Conclusion
Acknowledgments
Part III: Structure and Folding
Chapter Eight: SHAPE Analysis of Small RNAs and Riboswitches
Abstract
1 Theory
2 Equipment
3 Materials
4 Protocol
5 Step 1: RNA Folding and SHAPE Probing
6 Step 2: Primer Extension
7 Step 3: Capillary Electrophoresis
8 Step 4: Data Processing Using QuShape
9 Step 5: Data Processing and RNA Modeling
Acknowledgments
Chapter Nine: Experimental Approaches for Measuring pKa’s in RNA and DNA
Abstract
1 Introduction
2 Experimental Parameters for pH Titrations
3 RNA Cleavage Kinetics
4 Spectroscopic-Detected Methods
5 Perspective
Acknowledgments
Chapter Ten: Crystallographic Analysis of TPP Riboswitch Binding by Small-Molecule Ligands Discovered Through Fragment-Based Drug Discovery Approaches
Abstract
1 Introduction
2 Methods
3 Conclusions
Acknowledgments
Chapter Eleven: Methods for Using New Conceptual Tools and Parameters to Assess RNA Structure by Small-Angle X-Ray Scattering
Abstract
1 Introduction
2 Specialized Equipment
3 Preparation of the RNA for a SAXS Study
4 Interpretation of the X-Ray Scattering Curve
5 Case Studies
6 Multiphase Volumetric Modeling
7 Gold Labels and Comprehensive Conformations
8 Considerations
Acknowledgments
Part IV: Dynamics
Chapter Twelve: Use of 19F NMR Methods to Probe Conformational Heterogeneity and Dynamics of Exchange in Functional RNA Molecules
Abstract
1 Introduction
2 Methods
3 Conclusion and Remarks
Acknowledgments
Chapter Thirteen: Site-Directed Spin-Labeling Strategies and Electron Paramagnetic Resonance Spectroscopy for Large Riboswitches
Abstract
1 Techniques Used for Riboswitch Studies
2 Ligation Methods for SDSL of Large Riboswitches
3 CW-EPR Spectral Analysis of Riboswitches
Chapter Fourteen: Using sm-FRET and Denaturants to Reveal Folding Landscapes
Abstract
1 Introduction
2 Single-Molecule FRET: Technical Aspects
3 Riboswitch Structure and Biological Function
4 Combination of sm-FRET and Denaturants to Investigate Riboswitch Folding
5 Summary and Prospects
Chapter Fifteen: Riboswitch Structure and Dynamics by smFRET Microscopy
Abstract
1 Introduction
2 Methods
3 Practical Experimental Considerations
4 Data Analysis
5 Induced-Fit Versus Conformational Selection
6 Summary and Conclusions
Acknowledgments
Chapter Sixteen: Ribosome Structure and Dynamics by smFRET Microscopy
Abstract
1 Introduction
2 Overview of Ribosome Structure and Function
3 Methodology
4 Ribosome Dynamics
5 Data Acquisition
6 Building and Verifying Histograms, Normalization, Gaussian Fitting
7 Future Directions
Chapter Seventeen: Unraveling the Thermodynamics and Kinetics of RNA Assembly: Surface Plasmon Resonance, Isothermal Titration Calorimetry, and Circular Dichroism
Abstract
1 The RNA Folding Problem and Assembly of RNA Tertiary Structure
2 Practical Aspects of Biophysical Studies of RNA Assembly
3 Specific Measurements of Ion-Driven RNA Assembly
4 Concluding Remarks
Acknowledgments
Part V: Ligand Interactions
Chapter Eighteen: ITC Analysis of Ligand Binding to PreQ1 Riboswitches
Abstract
1 Introduction
2 Experimental Procedures for ITC
Acknowledgments
Chapter Nineteen: Facile Characterization of Aptamer Kinetic and Equilibrium Binding Properties Using Surface Plasmon Resonance
Abstract
1 Introduction
2 Materials
3 Sensor Surface Immobilization
4 Characterization of Aptamer Binding Properties
5 Conclusion
Acknowledgments
Chapter Twenty: The AdoCbl–Riboswitch Interaction Investigated by In-Line Probing and Surface Plasmon Resonance Spectroscopy (SPR)
Abstract
1 Introduction
2 In-Line Probing Experiments
3 SPR Spectroscopy
4 Conclusion
Acknowledgments
Chapter Twenty-One: Assessing RNA Interactions with Proteins by DRaCALA
Abstract
1 Introduction
2 DRaCALA-Based Detection of Protein–Ligand Interactions
3 Principle of DRaCALA
4 Determination of Fraction Bound by DRaCALA
5 Steps for Performing DRaCALA to Detect Protein Interaction With RNA
6 CsrA Binds Specifically to RsmY and RsmZ
7 Other Modifications of DRaCALA for RNA–Protein Interactions
Author Index
Subject Index
No. of pages: 570
Language: English
Edition: 1
Volume: 549
Published: November 21, 2014
Imprint: Academic Press
Hardback ISBN: 9780128011225
eBook ISBN: 9780128013359
DB
Donald H. Burke-Aguero
Affiliations and expertise
Department of Molecular Microbiology & Immunology and Department of Biochemistry, University of Missouri, USA
Read Riboswitch Discovery, Structure and Function on ScienceDirect