RNA helicases / edited by Eckhard Jankowsky.

Format:

Book

Contributor:

Jankowsky, Eckhard.

Series:

Methods in enzymology ; v. 511.

Methods in enzymology, 0076-6879 ; v. 511

Language:

English

Subjects (All):

Catalytic RNA.

Physical Description:

1 online resource (589 p.)

Edition:

1st ed.

Place of Publication:

Amsterdam ; Boston : Elsevier/Academic Press, 2012.

Language Note:

English

Summary:

This volume of Methods in Enzymology aims to provide a reference for the diverse, powerful tools used to analyze RNA helicases. The contributions in this volume cover the broad scope of methods in the research on these enzymes. Several chapters describe quantitative biophysical and biochemical approaches to study molecular mechanisms and conformational changes of RNA helicases. Further chapters cover structural analysis, examination of co-factor effects on several representative examples, and the analysis of cellular functions of select enzymes. Two chapters outline approaches to the analys

Contents:

Front Cover; RNA Helicases; Copyright; Contents; Contributors; Preface; Methods in Enzymology; Chapter One: Analysis of Duplex Unwinding by RNA Helicases Using Stopped-Flow Fluorescence Spectroscopy; 1 Introduction; 2 Experimental Considerations for Stopped-Flow Fluorescence Experiments; 2.1 Duplex concentration; 2.1.1 Strand annealing; 2.1.2 Strand exchange; 2.2 Verification of the correlation of fluorescence changes to duplex unwinding; 2.3 Background fluorescence and inner filter effects; 3 Substrate Design and Preparation; 3.1 RNA labeling approaches; 3.2 RNA labeled with Cy dyes

3.3 RNA labeled with 2-AP3.3.1 Preparation and purification of fluorescently labeled duplexes; 3.4 Assessing the impact of the fluorophore on the unwinding reaction; 4 Designing and Performing Stopped-Flow Fluorescence Unwinding Experiments; 4.1 Identification of optimal wavelengths for excitation and emission; 4.2 Pre-steady-state approaches to mechanistically characterize unwinding: Single and multiple cycle regimes; 4.2.1 The single cycle regime; 4.2.2 Protocol for single cycle unwinding reactions; 4.2.3 The multiple cycle regime; 4.2.4 Protocol for multiple cycle unwinding reactions

5 Data Fitting and Analysis5.1 Data fitting; 5.2 Determining unwinding rate constants; 5.3 Determining functional equilibrium constants; Acknowledgments; References; Chapter Two: Analyzing ATP Utilization by DEAD-Box RNA Helicases Using Kinetic and Equilibrium Methods; 1 Introduction; 2 Reagents and Equipment; 2.1 Solution conditions and temperature; 2.2 RNA substrate(s); 2.3 DEAD-box proteins; 2.4 ATP, ADP, and mant-labeled nucleotides; 2.5 UV-visible spectrophotometer; 2.6 Fluorimeter; 2.7 Stopped-flow apparatus; 2.8 Quench-flow apparatus

3 Important Considerations for Initial DBP Characterization3.1 DBP oligomeric state and stability/aggregation; 3.2 RNA substrate selection/design; 3.2.1 DBP-RNA binding specificity; 3.2.2 RNA-stimulated ATPase activity; 3.2.3 DBP-RNA binding affinity; 3.2.4 ATP-dependent RNA unwinding; 3.2.5 RNA substrate characteristics; 3.3 Measuring RNA binding affinity and stoichiometry; 3.3.1 Measuring the equilibrium RNA binding affinity in the absence of bound nucleotide and with saturating ADP...; 4 Steady-State ATPase Measurements; 4.1 Motivation for measuring DBP ATPase activity at steady state

4.2 Steady-state ATPase method: Real-time enzyme-coupled assay5 Transient Kinetic Analysis of the DBP ATPase Cycle; 5.1 RNA saturation in transient kinetics experiments; 5.2 Fluorescent-labeled ATP and ADP: Reporting on nucleotide binding/dissociation, hydrolysis, isomerization, and product rel; 5.3 Measuring the ATP hydrolysis and Pi release rates; 5.3.1 ATP hydrolysis measured using quench-flow; 5.3.2 Pi release measured using stopped-flow; 5.3.3 Pi release data fitting and analysis; 6 ATP Hydrolysis Reversibility Measured by Isotope Exchange; 7 RNA Unwinding Assays and ATPase Coupling

8 Kinetic Simulations

Notes:

Description based upon print version of record.

Includes bibliographical references and indexes.

ISBN:

9786613689658

9781280779268

1280779268

9780123972293

0123972299

OCLC:

802058453