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List of contributors xix
Abbreviations xxiii
Applications of chemically synthesized RNA 1 (36)
Michael J. Gait
David J. Earnshaw
Mark A. Farrow
Jan H. Fogg
Richard L. Grenfell
Nikolai A. Naryshkin
Terence V. Smith
Basic methods of synthesis and purification 1 (11)
of oligoribonucleotides
Introduction 1 (1)
Assembly of oligoribonucleotides 2 (3)
Deprotection of oligoribonucleotides 5 (3)
Purification of oligoribonucleotides 8 (4)
Incorporation of modified ribonucleotides 12 (6)
Base analogues 12 (3)
Ribose analogues 15 (1)
Phosphate analogues 16 (2)
Analysis of oligoribonucleotides and 18 (5)
analogues
Capillary electrophoresis 18 (2)
MALDI-TOF mass spectrometry 20 (1)
Analysis of oligoribonucleotides by 21 (2)
enzymatic digestion and reversed-phase
HPLC
Synthetic RNA duplex models for HIV-1 Tat 23 (6)
protein interaction
Competition gel retardation assays 23 (4)
Interference filter binding assays 27 (2)
Cross-linking of peptides and proteins to 29 (8)
modified synthetic RNA
Acknowledgements 33 (1)
References 33 (4)
Preparation of RNA:protein complexes for 37 (38)
X-ray crystallography and NMR
Stephen R. Price
Chris Oubridge
Gabriele Varani
Kiyoshi Nagai
Introduction 37 (1)
Preparation of T7 RNA polymerase 38 (2)
In vitro transcription using 40 (11)
oligonucleotide or plasmid templates
RNA synthesis by run-off transcription 40 (3)
using oligonucleotide templates
In vitro transcription using plasmid 43 (8)
templates
Gel purification of transcribed RNA 51 (7)
Casting gels for preparative RNA 52 (1)
purification
Extraction of RNA from polyacrylamide gels 53 (2)
Desalting RNA 55 (3)
Concentration of RNA 58 (1)
Preparation of isotopically labelled RNA 58 (8)
for NMR
Purification of isotopically labelled RNA 59 (7)
from bacterial sources
Preparation of RNA:protein complexes for 66 (9)
crystallization and NMR analysis
Checking purity of RNA 66 (3)
Reconstituting the RNA:protein complex 69 (2)
Crystallization strategy of RNA:protein 71 (1)
complexes
Acknowledgements 72 (1)
References 72 (3)
Uses of site-specifically modified RNAs 75 (34)
constructed by RNA ligation
Melissa J. Moore
Charles C. Query
Introduction 75 (1)
Review of methods for introducing modified 75 (3)
nucleotides into small RNAs
Chemical synthesis 77 (1)
Transcription with phage polymerases 77 (1)
Coupling chemical synthesis to 78 (1)
transcription: dinucleotide primers
Synthesis of long RNAs by transcription 78 (4)
Techniques for RNA ligation 82 (8)
T4 RNA ligase 83 (1)
T4 DNA ligase 83 (7)
Chemical ligation 90 (1)
Examples of cross-linking proteins to 90 (15)
site-specifically modified RNAs
Photocross-linking proteins to RNA 90 (2)
Path labelling 92 (2)
Using T4 DNA ligase to incorporate single 94 (4)
labels at internal sites in RNAs
Convertible nucleotides 98 (4)
Other photocross-linkable nucleotides 102 (1)
Determining cross-link specificity 103 (2)
Additional uses for RNA ligation 105 (4)
References 106 (3)
The electrophoretic mobility shift assay for 109 (28)
RNA binding proteins
Douglas L. Black
Raymond Chan
Hosung Min
Jiwu Wang
Leslie Bell
Introduction 109 (2)
Principle of electrophoretic mobility 109 (1)
shift assay (EMSA)
General considerations 109 (2)
Starting material: RNA and protein 111 (3)
Preparation of mammalian nuclear extracts 111 (2)
Preparing radiolabelled RNA for binding 113 (1)
studies
Large scale synthesis of unlabelled RNA 114 (1)
EMSA for protein:RNA interactions 114 (6)
Binding conditions for the DCS complex 114 (1)
Separation of RNA:protein complexes by 115 (1)
native gel electrophoresis
Optimizing the binding reaction 116 (1)
The stability of an RNA:protein complex 117 (2)
within the gel
Measuring the sequence specificity of 119 (1)
binding
Characterization of the proteins within a 120 (4)
complex
UV cross-linking within a gel isolated 120 (3)
complex
Antibody supershift and disruption 123 (1)
experiments
EMSA as an assay for fractionation 124 (1)
RNA binding analysis of purified proteins 124 (13)
EMSA with purified protein 124 (1)
Filter binding assay 125 (1)
Establishing the binding conditions 126 (2)
Determining the dissociation constant 128 (2)
Multiple binding sites and co-operativity 130 (5)
References 135 (2)
Affinity methods for isolating RNA binding 137 (24)
proteins
Ann Kaminski
Dirk H. Ostareck
Nancy M. Standart
Richard J. Jackson
Introduction 137 (4)
Scope of the chapter 137 (1)
Overview of methods for affinity 138 (1)
purification of RNA binding protiens
Protein purification prior to affinity 139 (1)
chromatography
Suggestions with regard to prioritizing 140 (1)
approaches
Large scale transcription reactions using 141 (2)
bacteriophage RNA polymerases
General recommendations for running RNA 143 (1)
affinity columns
Coupling RNA to CNBr-activated Sepharose 144 (4)
Use of affinity columns made by coupling 146 (2)
RNA to CNBr-activated Sepharose
The use of poly(A) tailed RNA affinity 148 (2)
substrates
Biotinylated RNA as an affinity matrix 150 (6)
Overview of methods for preparing 150 (1)
biotinylated RNA affinity substrates
Modification of RNA by direct 151 (2)
incorporation of biotinylated NTP
Two-step modification of RNA by 153 (1)
incorporation of an amine-modified NTP,
and subsequent biotinylation with
biotinylated easter
Synthetic biotinylated 154 (1)
oligoribonucleotides
Recovery of RNA binding proteins from the 155 (1)
biotinylated RNA affinity matrix
Other affinity columns for selectively 156 (1)
removing specific RNA binding proteins
The use of affinity columns for selectively 157 (4)
removing specific RNA binding proteins from
extracts
Acknowledgements 158 (1)
References 158 (3)
Synthentic lethal/enhancer screening to 161 (22)
identify snRNA:protein and protein:protein
interactions in yeast pre-mRNA splicing
Francoise Stutz
Jie Tang
Michael Rosbash
Introduction 161 (4)
U1 snRNA enhancer screen using a viability 165 (10)
assay
General reagents and strains 165 (1)
Protocols 166 (5)
Results and discussion of U1 snRNA 171 (4)
enhancer screen
Identification of synthetic lethal 175 (6)
mutations using the red/white sectoring
assay
General reagents and strains 176 (1)
Protocols 177 (3)
Results and discussion of MUD2 synthetic 180 (1)
lethal screen
Concluding remarks 181 (2)
References 181 (2)
Detecting RNA:protein interactions and 183 (12)
isolating cDNA clones by Northwestern
screening
Jeffrey Wilusz
Introduction 183 (2)
General considerations for Northwestern 183 (1)
detection of RNA:protein interactions
Probability of success 184 (1)
Preparation of RNA probes 185 (2)
Northwestern blots to detect RNA blinding 187 (2)
proteins
Northwestern screening of cDNA libraries 189 (6)
References 193 (2)
A three-hybrid system to detect and analyse 195 (22)
RNA:protein interactions in vivo
Beilin Zhang
Brian Kraemer
Dhruba Sengupta
Stanley Fields
Marvin Wickens
Introduction 195 (1)
Strategy of the three-hybrid system 196 (1)
General considerations 197 (2)
Potential applications 199 (1)
Hybrid RNA molecules: general features 200 (4)
Constructing plasmids that encode hybrid 201 (3)
RNA molecules
Reporter strain 204 (1)
The activation domain plasmid 205 (1)
Analysing known RNA:protein interactions 205 (5)
Screening for new proteins with a known RNA 210 (5)
sequence: general strategy and
considerations
Perspective 215 (2)
Acknowledgements 215 (1)
References 215 (2)
In vivo selection of specific RNA binding 217 (20)
polypeptides using a transcription
anti-termination reporter assay
Kazuo Harada
Alan D. Frankel
Introduction 217 (1)
Detection of RNA:polypeptide interactions 218 (4)
using the bacterial transcription
anti-termination system
Strategies in the design of combinatorial 222 (7)
libraries
Library design 222 (3)
Preparation of combinatorial 225 (4)
oligonucleotide cassettes
Selection of novel peptides that bind to 229 (3)
specific RNAs
Selection from randomized libraries 229 (2)
Selection from doped libraries 231 (1)
Assessing activity 232 (3)
β-Galactosidase solution assay 232 (1)
In vitro RNA binding gel shift assay 233 (2)
Summary and future directions 235 (2)
References 235 (2)
Footprinting and modification-interference 237 (18)
analysis of binding sites on RNA
Chuck Merryman
Harry F. Noller
Introduction 237 (1)
Experimental approach 238 (2)
General considerations 238 (1)
Footprinting 238 (1)
Modification-interference 239 (1)
Chemical probing of RNA 240 (6)
Chemicalprobes 240 (1)
Probing conditions for footprinting 241 (1)
experiments
Probing conditions for 241 (1)
modification-interference experiments
Protocols for chemical probing 242 (4)
Primer extension 246 (3)
Interpretation 249 (6)
Footprinting 251 (1)
Modification-interference 251 (1)
Concluding remarks 251 (2)
References 253 (2)
Analysis of RNA:protein and RNA:RNA contacts 255 (30)
between ribosomes and functional ligands
using site-directed cross-linking techniques
Jutta Rinke-Appel
Richard Brimacombe
Introduction 255 (1)
General considerations 256 (3)
Insertion of photoreactive agents into 259 (9)
ribosomal ligands
Preparation of mRNA analogues containing 259 (5)
4-thioU by T7 polymerase
The use of diazirine or azide reagents 264 (2)
for specific reaction with tRNA molecules
at naturally modified positions
Modification of amino acids with TDB for 266 (2)
in situ peptide biosynthesis
Formation of ribosomal complexes for 268 (6)
cross-linking and isolation of cross-linked
material
Preparation of 70S ribosomes 268 (4)
Separation of cross-linked ribosomal 272 (1)
components by sucrose gradient
centrifugation
Affinity chromatography on oligo(dT) 273 (1)
cellulose
Cross-linking yield 274 (1)
Analysis of cross-link sites within rRNA 274 (3)
Partial digestions of rRNA by 274 (3)
ribonuclease H in the presence of
oligodeoxynucleotides
Identification of cross-link sites within 277 (1)
rRNA by primer extension
Identification of cross-linked proteins 277 (2)
Identification

RNA-Protein Interactions: A Practical Approach provides a clear and comprehensive guide to the many experimental procedures used in studying RNA-protein interactions. The approaches covered range from those initially used to detect a novel RNA-protein interaction to various biochemical and genetic approaches, and from in-depth methods for analyzing the structural basis of the interaction to methods for purifying and cloning RNA binding proteins. The volume contains a number of procedures that have not previously been covered in this type of manual, including the production of site-specifically modified RNAs by enzymatic and chemical methods and in vivo screening for novel RNA-protein interactions in yeast and E. coli. This informative book will be a valuable aid to laboratory workers in biochemistry and molecular biology, as well as graduate students and postdoctoral and senior scientists, whose research encompasses this field.