Riboswitches - Methods and Protocols

The transfer of hereditary information from genes to proteins is one of the essential pr- esses in all living organisms on our planet. Some genes are expressed without modu- tion throughout the life of a cell, while many others require various degrees of control to precisely balance cellular metabolism with environmental conditions. For many years, researchers attributed this regulatory function to protein molecules, which can direct gene expression at multiple levels, in response to various input signals, and with different degrees of selectivity. Even when the control of gene expression was achieved via direct interactions between proteins and mRNAs, the active role was routinely assigned to p- teins, while RNAs were considered merely as recipient molecules. The discovery of RNA interference and multiple bacterial regulatory RNAs caused a shift from the perception of proteins as the predominant regulators of gene expression to the acknowledgement of the importance of RNAs in many regulatory circuits. Such a viewpoint received strong support several years ago after the discovery of riboswitches and related RNA sensors - mRNA regions capable of alternating their conformations in response to the presence of cellular metabolites and other physical or chemical cues. These classes of RNA pass on cellular and environmental information directly to transcription or translation machinery without the assistance of proteins. The riboswitches are commonly defined as evolutionarily conserved mRNA regions capable of specific binding to metabolite molecules, and, as a result, adopting a particular RNA conformation that modulates gene expression.

List of contents

Predicting Riboswitch Regulation on a Genomic Scale.- Enzymatic Ligation Strategies for the Preparation of Purine Riboswitches with Site-Specific Chemical Modifications.- Application of Fluorescent Measurements for Characterization of Riboswitch-Ligand Interactions.- Transcriptional Approaches to Riboswitch Studies.- Kinetics of Riboswitch Regulation Studied By In Vitro Transcription.- Molecular Basis of RNA-Mediated Gene Regulation on the Adenine Riboswitch by Single-Molecule Approaches.- Methods for Analysis of Ligand-Induced RNA Conformational Changes.- Monitoring RNA-Ligand Interactions Using Isothermal Titration Calorimetry.- Preparation and Crystallization of Riboswitch-Ligand Complexes.- Crystallization of the glmS Ribozyme-Riboswitch.- Riboswitch Conformations Revealed by Small-Angle X-Ray Scattering.- Time-Resolved NMR Spectroscopy: Ligand-Induced Refolding of Riboswitches.- Analysis of the RNA Backbone: Structural Analysis of Riboswitches by In-Line Probing and Selective 2'-Hydroxyl Acylation and Primer Extension.- Identification of Metabolite-Riboswitch Interactions Using Nucleotide Analog Interference Mapping and Suppression.- RNA-Dependent RNA Switches in Bacteria.- Probing mRNA Structure and sRNA-mRNA Interactions in Bacteria Using Enzymes and Lead(II).- Structural Probing of RNA Thermosensors.- Ribosomal Initiation Complexes Probed by Toeprinting and Effect of trans-Acting Translational Regulators in Bacteria.- Isolation and Characterization of the Heat Shock RNA 1.- Analysis of tRNA-Directed Transcription Antitermination in the T Box System In Vivo.- In Vitro Selection of Conformational Probes for Riboswitches.- A Green Fluorescent Protein (GFP)-Based Plasmid System to Study Post-Transcriptional Control of Gene Expression In Vivo.-High-Throughput Screens to Discover Synthetic Riboswitches.- A Mammalian Cell-Based Assay for Screening Inhibitors of RNA Cleavage.- In Vitro Selection of glmS Ribozymes.

Summary

The transfer of hereditary information from genes to proteins is one of the essential pr- esses in all living organisms on our planet. Some genes are expressed without modu- tion throughout the life of a cell, while many others require various degrees of control to precisely balance cellular metabolism with environmental conditions. For many years, researchers attributed this regulatory function to protein molecules, which can direct gene expression at multiple levels, in response to various input signals, and with different degrees of selectivity. Even when the control of gene expression was achieved via direct interactions between proteins and mRNAs, the active role was routinely assigned to p- teins, while RNAs were considered merely as recipient molecules. The discovery of RNA interference and multiple bacterial regulatory RNAs caused a shift from the perception of proteins as the predominant regulators of gene expression to the acknowledgement of the importance of RNAs in many regulatory circuits. Such a viewpoint received strong support several years ago after the discovery of riboswitches and related RNA sensors – mRNA regions capable of alternating their conformations in response to the presence of cellular metabolites and other physical or chemical cues. These classes of RNA pass on cellular and environmental information directly to transcription or translation machinery without the assistance of proteins. The riboswitches are commonly defined as evolutionarily conserved mRNA regions capable of specific binding to metabolite molecules, and, as a result, adopting a particular RNA conformation that modulates gene expression.

Additional text

From the reviews:

"Riboswitches are among the latest additions to the growing field of RNA-based gene expression control systems found in both prokaryotic and eukaryotic cells. … useful in various aspects of work on riboswitches and other regulatory RNAs. … It also refers to pertinent and up-to-date literature. … The reviewed book can be strongly recommended to RNA researchers with different levels of expertise, as well as to those who learn or teach in this field." (Joanna Ciesla, Acta Biochimica Polonica, Vol. 56 (3), 2009)

“This book is a good collection of ‘Methods and Protocols’ for studying … riboswitches as well as some other RNAs involved in gene expression. … Readers are introduced to a broad range of RNA methodologies from basic to applied in this book. I … recommend the book for researchers who work in the area of riboswitches, but also for other scientists who are involved in RNA-relevant studies.” (Atsushi Ogawa, ChemBioChem, Vol. 10, 2009)

Report

From the reviews:
"Riboswitches are among the latest additions to the growing field of RNA-based gene expression control systems found in both prokaryotic and eukaryotic cells. ... useful in various aspects of work on riboswitches and other regulatory RNAs. ... It also refers to pertinent and up-to-date literature. ... The reviewed book can be strongly recommended to RNA researchers with different levels of expertise, as well as to those who learn or teach in this field." (Joanna Ciesla, Acta Biochimica Polonica, Vol. 56 (3), 2009)
"This book is a good collection of 'Methods and Protocols' for studying ... riboswitches as well as some other RNAs involved in gene expression. ... Readers are introduced to a broad range of RNA methodologies from basic to applied in this book. I ... recommend the book for researchers who work in the area of riboswitches, but also for other scientists who are involved in RNA-relevant studies." (Atsushi Ogawa, ChemBioChem, Vol. 10, 2009)

Product details

• Assisted by: Alexande Serganov (Editor), Alexander Serganov (Editor)
• Publisher: Springer, Berlin
• Languages: English
• Product format: Paperback / Softback
• Released: 12.05.2011
• EAN: 9781617379475
• ISBN: 978-1-61737-947-5
• No. of pages: 366
• Weight: 782 g
• Illustrations: XI, 366 p. 65 illus.
• Series: Methods in Molecular Biology; Methods in Molecular Biology
• Subjects: Natural sciences, medicine, IT, technology > Biology > Biochemistry, biophysics; B, Life Sciences, biochemistry, Biomedical and Life Sciences, Medical Genetics, Biochemistry, general, Genetics (non-medical), Gene Expression, Nucleic acids, Nucleic Acid Chemistry, Engineering applications of bio-materials