RNA'Protein Interaction Protocols

1. Front Matter

   Pages i-xix

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2. Labeling and Purification of RNA Synthesized by In Vitro Transcription

   • Paul A. Clarke

   Pages 1-10

3. Joining RNA Molecules with T4 DNA Ligase

   • Melissa J. Moore

   Pages 11-19

4. RNA-Protein Crosslinking with Photoreactive Nucleotide Analogs

   • Michelle M. Hanna, Lori Bentsen, Michael Lucido, Archana Sapre

   Pages 21-33

5. The Methylene Blue Mediated Photocrosslinking Method for Dectection of Proteins that Interact with Double-Stranded RNA

   • Zhi-Ren Liu, Christopher W.J. Smith

   Pages 35-47

6. Probing RNA-Protein Interactions by Psoralen Photocrosslinking

   • Zhuying Wang, Tariq M. Rana

   Pages 49-62

7. Identification and Sequence Analysis of RNA-Protein Contact Sites by N-Terminal Sequencing and MALDI-MS

   • Bernd Thiede, Henning Urlaub, Helga Neubauer, Brigitte Wittmann-Liebold

   Pages 63-72

8. RNA Footprinting and Modification Interference Analysis

   • Paul A. Clarke

   Pages 73-91

9. Oligonucleotide-Targeted RNase H Protection Analysis of RNA-Protein Complexes

   • Arthur Günzl, Albrecht Bindereif

   Pages 93-103

10. Nitrocellulose Filter Binding for Determination of Dissociation Constants

    • Kathleen B. Hall, James K. Kranz

    Pages 105-114

11. Measuring Equilibrium and Kinetic Constants Using Gel Retardation Assays

    • David R. Setzer

    Pages 115-128

12. PACE Analysis of RNA-Peptide Interactions

    • Christopher D. Cilley, James R. Williamson

    Pages 129-141

13. Detection of Nucleic Acid Interactions Using Surface Plasmon Resonance

    • Robert J. Crouch, Makoto Wakasa, Mitsuru Haruki

    Pages 143-160

14. An Escherichia coli-Based Genetic Strategy for Characterizing RNA Binding Proteins

    • Chaitanya Jain

    Pages 161-175

15. Screening RNA-Binding Libraries Using a Bacterial Transcription Antitermination Assay

    • Kazuo Harada, Alan D. Frankel

    Pages 177-187

16. In Vitro Genetic Analysis of RNA-Binding Proteins Using Phage Display

    • Ite A. Laird-Offringa

    Pages 189-216

17. In Vitro Selection of Aptamers from RNA Libraries

    • Daniel J. Kenan, Jack D. Keene

    Pages 217-231

18. Identification of Specific Protein-RNA Target Sites Using Libraries of Natural Sequences

    • Lucy G. Andrews, Jack D. Keene

    Pages 233-244

19. Northwestern Screening of Expression Libraries

    • Paramjeet S. Bagga, Jeffrey Wilusz

    Pages 245-256

20. Screening Expression Libraries with Solution-Based Assay

    • Philippa J. Webster, Paul M. Macdonald

    Pages 257-264

The molecular characterization of RNA and its interactions with proteins is an important and exciting area of current research. Organisms utilize a variety of RNA–protein interactions to regulate the expression of their genes. This is particularly true for eukaryotes, since newly synthesized messenger RNA must be extensively modified and transported to the cytoplasm before it can be used for protein synthesis. The realization that posttranscriptional processes are critical components of gene regulation has sparked an explosion of interest in both stable ribonucleoprotein (RNP) complexes and transient RNA–protein interactions. RNA is conformationally flexible and can adopt complex structures that provide diverse surfaces for interactions with proteins. The fact that short RNA molecules (aptamers; see Chapter 16) can be selected to bind many different types of molecules is evidence of the structural variability of RNA. RNA molecules are rarely entirely single- or double-stranded, but usually contain multiple short duplexes interrupted by single-stranded loops and bulges; in some RNAs, such as tRNAs, the short duplexes stack on each other. Further variability is generated by the presence of non-Watson-Crick base pairs, modified nucleotides, and more complex structures, such as pseudoknots and triple-strand interactions.

"This manual is recommended to investigators with experience in molecular biological research, wishing to become introduced into the field of RNA-protein interactions. This book will also be a valuable source of reference for scientists needing general information about the theoretical basis and current methodological approaches to investigating ribonucleoprotein complexes."-International Journal of Medical Microbiology

"The coverage of the book is broad and reasonably comprehensive, with chapters arranged in a coherent sequence....Many of the authors are well-known, leading researchers in their field, and describe key contemporary techniques. In these respects the coverage is authoritative and likely to remain relevant for some years, before being overtaken by new technologies and methods...represents value for money."-Cell Biology International

"This is a book which any scientist involved in any kind of cellular and molecular biology will find profitable for his experimental investigations since RNA and proteins are unavoidable matters to solve any problem in Life Sciences. Highly skilled experimentalists provide a comprehensive series of commonly used, as well as specialized techniques for analyzing how proteins and RNA interact. Richly detailed and readily reproducible, these methods enable researchers to analyze the structural details of an RNA-protein interaction, to determine in detail what parts of the protein and RNA are in close contact, and to isolate RNP complexes from cells." - Cellular and Molecular Biology

• Uniformed Services University of the Health Sciences, Bethesda

  Susan R. Haynes

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