Translation initiation : reconstituted systems and biophysical methods / edited by Jon Lorsch.

Format:

Book

Contributor:

Lorsch, Jon.

Series:

Methods in enzymology ; v. 430.

Methods in enzymology, 0076-6879 ; v. 430

Language:

English

Subjects (All):

Genetic translation.

Genetic regulation.

Physical Description:

1 online resource (510 p.)

Place of Publication:

Amsterdam ; London : Elsevier, c2007.

Language Note:

English

Summary:

For over fifty years the Methods in Enzymology series has been the critically aclaimed laboratory standard and one of the most respected publications in the field of biochemistry. The highly relevant material makes it an essential publication for researchers in all fields of life and related sciences. This volume, the second of three on the topic of Translation Initiation includes articles written by leaders in the field.

Contents:

Front Cover; Translation Initiation: Reconstituted Systems and Biophysical Methods; Copyright Page; Contents; Contributors; Preface; Volume in Series; Chapter 1: Transient Kinetics, Fluorescence, and FRET in Studies of Initiation of Translation in Bacteria; 1. Introduction; 2. Experimental Outline; 3. Materials; 3.1. Stock solutions; 3.2. Reagents; 4. Experimental Procedures; 4.1. Preparation and fluorescence labeling of ribosomes and ribosomal subunits; 4.2. Zonal centrifugation; 4.3. Preparation and fluorescence labeling of initiator fMet-tRNAfMet

4.4. Preparation of fluorescence-labeled mRNA4.5. Initiation factors; 5. Rapid Kinetic Measurements; 5.1. Measuring fluorescence intensities and FRET changes in stopped flow; 6. Quench-Flow Measurements; 7. GTPase Activity; 8. Dipeptide Formation; 9. Applications of the Method; 10. Binding of fMet-tRNA to the 30S Subunit; 11. FRET to IF3; 12. Nucleotide Binding to IF2; 13. Subunit Joining; Acknowledgments; References; Chapter 2: Binding of mRNA to the Bacterial Translation Initiation Complex; 1. Introduction; 2. Experimental Procedures; 2.1. Design of model mRNAs

2.2. Synthesis of model mRNAs2.3. Deprotection; 2.4. Thermal melting analysis; 2.5. Labeling and purification of mRNA; 2.6. Labeling schemes; 2.7. Fluorophores; 2.8. Isolation of the 30S subunit and protein purification; 2.9. Purification of aminoacylated initiator tRNA; 2.10. Initiation complex assembly; 2.11. mRNA dissociation; 2.12. Steady-state fluorescence measurements; 2.13. Kinetic association and dissociation experiments; 2.14. Determination of equilibrium binding constants; Acknowledgments; References

Chapter 3: Real-Time Dynamics of Ribosome-Ligand Interaction by Time-Resolved Chemical Probing Methods1. Introduction; 2. General Strategy; 2.1. Buffers and mixtures; 2.2. Analysis of probing reactions; 3. Time-Resolved Chemical Probing with DMS; 3.1. Validation of time-resolved chemical probing with DMS; 3.2. Procedure for chemical probing with DMS; 3.3. Example of time-resolved chemical probing with DMS; 4. Time-Resolved Probing with ONOOK; 4.1. Validation of time-resolved probing with ONOOK; 4.2. Preparation of ONOOK; 4.3. Procedure for time-resolved probing with ONOOK

4.4. Example of time-resolved probing with ONOOK5. Time-Resolved Chemical Probing with Fe(II)-EDTA; 5.1. Validation of time-resolved probing with Fe(II)-EDTA; 5.2. Procedure for time-resolved. probing with Fe(II)-EDTA; 5.3. Example of time-resolved probing with Fe(II)-EDTA; Acknowledgments; References; Chapter 4: Overexpression and Purification of Mammalian Mitochondrial Translational Initiation Factor 2 and Initiation Factor 3; 1. Introduction; 2. Materials; 2.1. Growth media; 2.2. Common buffers; 2.3. Buffers for IF2mt; 2.4. Buffers for IF3mt; 2.5. Reagents; 2.6. Preparation of reagents

2.7. The high-performance liquid chromatography (HPLC) system

Notes:

Description based upon print version of record.

Includes bibliographical references and indexes.

ISBN:

9786611049683

9781281049681

1281049689

9780080553184

0080553184

OCLC:

476111962