Riboswitches

This book provides comprehensive, up-to-date coverage of many methods used to study riboswitches and other RNAs involved in gene expression control. It focuses on mRNAs responding to small organic molecules and offers an expanded definition of a riboswitch.

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.

A comprehensive and timely reference volume for basic and applied research on regulatory RNAs Covers a wide range of assay formats and systems from the computerized prediction of regulatory RNA elements to their detailed structure-function characterization Focuses on both natural and synthetic RNA molecules Provides step-by-step protocols of rigorously tested classical and cutting-edge in vivo and in vitro methods Presents techniques easily adaptable for experiments with any RNA species Includes supplementary material: sn.pub/extras

Klappentext

The revolutionary discoveries of RNA interference and bacterial regulatory RNAs led to further breakthroughs such as the identification 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. In Riboswitches: Methods and Protocols, expert researchers provide comprehensive and up-to-date coverage of various methods used to study riboswitches and other RNAs involved in gene expression control. Examining biochemical and modern biophysical techniques, the volume focuses on mRNAs responding to small organic molecules but expands the definition of a riboswitch to incorporate classes of RNA that undergo conformational transitions in response to other stimuli in order to control the expression of genes. Written in the highly successful Methods in Molecular Biology™ series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible protocols, and notes on troubleshooting and avoiding known pitfalls.

Cutting-edge and authoritative, Riboswitches: Methods and Protocols promises to inspire both novices and experts working in a wide range of contemporary biological areas who wish to further develop their RNA methodology.

Inhalt
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 RiboswitchLigand 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 RNALigand Interactions Using Isothermal Titration Calorimetry.- Preparation and Crystallization of RiboswitchLigand 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 MetaboliteRiboswitch Interactions Using Nucleotide Analog Interference Mapping and Suppression.- RNA-Dependent RNA Switches in Bacteria.- Probing mRNA Structure and sRNAmRNA 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.