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 first of three on the topic of Translation Initiation includes articles written by leaders in the field.
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METHODS IN ENZYMOLOGY Editors-in-Chief
JOHN N. ABELSON AND MELVIN I. SIMON Division of Biology California Institute of Technology Pasadena, California Founding Editors
SIDNEY P. COLOWICK AND NATHAN O. KAPLAN
CONTENTS
Contributors Preface Volumes in Series
1. Transient Kinetics, Fluorescence, and FRET in Studies of Initiation of Translation in Bacteria
xiii xix xxi
1
Pohl Milon, Andrey L. Konevega, Frank Peske, Attilio Fabbretti, Claudio O. Gualerzi, and Marina V. Rodnina 1. Introduction 2. Experimental Outline 3. Materials 4. Experimental Procedures 5. Rapid Kinetic Measurements 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
2. Binding of mRNA to the Bacterial Translation Initiation Complex
2 5 9 10 20 21 22 22 23 23 23 25 25 26 26
31
Sean M. Studer and Simpson Joseph 1. Introduction 2. Experimental Procedures Acknowledgments References
32 33 43 43
v
vi
Contents
3. Real-Time Dynamics of Ribosome-Ligand Interaction by Time-Resolved Chemical Probing Methods
45
Attilio Fabbretti, Pohl Milon, Anna Maria Giuliodori, Claudio O. Gualerzi, and Cynthia L. Pon 1. Introduction 2. General Strategy