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Ribozymes and RNA Catalysis: Introduction and Primer | |
What are Ribozymes? | p. 1 |
What is the Role of Ribozymes in Cells? | p. 1 |
Ribozymes Bring about Significant Rate Enhancements | p. 4 |
Why Study Ribozymes? | p. 4 |
Folding RNA into the Active Conformation | p. 5 |
The Catalytic Resources of RNA - Making a Lot of a Little | p. 6 |
Mechanisms and Catalytic Strategies of Ribozymes | p. 7 |
Impact of New Methodologies to Study Ribozymes | p. 8 |
Finally | p. 8 |
References | p. 8 |
Proton Transfer in Ribozyme Catalysis | |
Scope of Chapter and Rationale | p. 11 |
Overview of Proton Transfer Chemistry | p. 12 |
General Considerations for Proton Transfer in RNA Enzymes | p. 17 |
Classes of Protonation Sites in RNA | p. 17 |
Driving Forces for pKa Shifting in RNA | p. 18 |
Quantitative Contributions of Proton Transfer to RNA Catalysis | p. 19 |
Proton Transfer in Small Ribozymes: Five Case Studies | p. 20 |
Why Small Ribozymes? | p. 20 |
Proton Transfer in the Hepatitis Delta Virus Ribozyme | p. 22 |
Proton Transfer in the Hairpin Ribozyme | p. 27 |
Proton Transfer in the Hammerhead Ribozyme | p. 28 |
Proton Transfer in the VS Ribozyme | p. 29 |
Proton Transfer in the glmS Ribozyme | p. 30 |
Conclusion and Perspectives | p. 31 |
Acknowledgement | p. 32 |
References | p. 32 |
Finding the Hammerhead Ribozyme Active Site | |
Introduction | p. 37 |
Background | p. 38 |
Experimental Data | p. 40 |
Mechanistic Hypothesis Leads to Identification and Functional Test of Active Site Components | p. 40 |
Structural Hypothesis - Large-scale Conformational Changes are Required for Catalysis | p. 41 |
Molecular Modeling of a Hammerhead Active Fold that Satisfies Structural and Biochemical Constraints | p. 43 |
Current Status and Future Prospects | p. 46 |
Acknowledgements | p. 46 |
References | p. 46 |
Hammerhead Ribozyme Crystal Structures and Catalysis | |
Introduction | p. 48 |
A Catalytic RNA Prototype | p. 49 |
A Small Ribozyme | p. 49 |
Chemistry of Phosphodiester Bond Isomerization | p. 50 |
Hammerhead Ribozyme Structure | p. 51 |
Catalysis in the Crystal | p. 53 |
Making Movies from Crystallographic Snapshots | p. 53 |
An Ever-growing List of Concerns | p. 55 |
Occam's Razor Can Slit Your Throat | p. 56 |
Structure of a Full-length Hammerhead Ribozyme | p. 57 |
Do the Minimal and Full-length Hammerhead Crystal Structures have Anything in Common? | p. 59 |
How Does the Minimal Hammerhead Work? | p. 60 |
A Movie Sequel with a Happy Ending | p. 61 |
Concluding Remarks | p. 62 |
Acknowledgements | p. 62 |
References | p. 62 |
The Hairpin and Varkud Satellite Ribozymes | |
Nucleolytic Ribozymes | p. 66 |
Hairpin Ribozyme | p. 66 |
Structure of the Hairpin Ribozyme | p. 67 |
Metal Ion-dependent Folding of the Hairpin Ribozyme | p. 69 |
Observing the Cleavage and Ligation Activities of the Hairpin Ribozyme | p. 71 |
Mechanism of the Hairpin Ribozyme | p. 73 |
VS Ribozyme | p. 76 |
Structure of the VS Ribozyme | p. 77 |
Structure of the Substrate | p. 80 |
Location of the Substrate | p. 80 |
Active Site of the VS Ribozyme | p. 82 |
Candidate Catalytic Nucleobases | p. 82 |
Mechanism of the VS Ribozyme | p. 84 |
Some Striking Similarities between the Hairpin and VS Ribozymes | p. 88 |
Acknowledgements | p. 88 |
References | p. 88 |
Catalytic Mechanism of the HDV Ribozyme | |
Introduction | p. 92 |
Hepatitis Delta Virus Biology | p. 92 |
Cleavage Reactions of Small Ribozymes | p. 93 |
HDV Ribozyme Structure | p. 95 |
Determination of Crystal Structures | p. 95 |
Structure Overview | p. 97 |
Active Site | p. 97 |
Catalytic Strategies for RNA Cleavage | p. 99 |
The Active Site Nucleobase: C75 | p. 100 |
Exogenous Base Rescue Reactions | p. 101 |
Role of C75 in HDV Catalysis | p. 103 |
Resolving the Kinetic Ambiguity | p. 105 |
Reaction in the Absence of Divalent Cations | p. 105 |
Sulfur Substitution of the Leaving Group | p. 106 |
Metal Ions in the HDV Ribozyme | p. 108 |
Structural Metal Ions | p. 108 |
Catalytic Metal Ions | p. 111 |
Contributions of Non-active-site Structures to Catalysis | p. 112 |
Dynamics in HDV Function | p. 113 |
Varieties of Experimental Systems | p. 115 |
Models for HDV Catalysis | p. 117 |
Conclusion | p. 119 |
Acknowledgements | p. 120 |
References | p. 120 |
Mammalian Self-Cleaving Ribozymes | |
Introduction | p. 123 |
General Features of Small Self-cleaving Sequences | p. 124 |
Genome-wide Selection of Self-cleaving Ribozymes | p. 124 |
CPEB3 Ribozyme | p. 125 |
Expression of the CPEB3 Ribozyme | p. 126 |
Structural Features of the CPEB3 and HDV Ribozymes | p. 127 |
Linkage of HDV to the Human Transcriptome | p. 129 |
Possible Biological Roles of Self-cleaving Ribozymes | p. 130 |
Closing Remarks | p. 131 |
References | p. 131 |
The Structure and Action of glmS Ribozymes | |
Introduction | p. 134 |
Biochemical Characteristics of glmS Ribozymes | p. 136 |
Divalent Metal Ions Support Structure and Not Chemistry | p. 136 |
Ligand Specificity of glmS Ribozymes | p. 137 |
Evidence for a Coenzyme Role for GlcN6P | p. 139 |
Atomic-resolution Structure of glmS Ribozymes | p. 141 |
Secondary and Tertiary Structures of glmS Ribozymes | p. 141 |
Metabolite Recognition by glmS Ribozymes | p. 143 |
Mechanism of glmS Ribozyme Self-cleavage | p. 145 |
Can glmS Ribozymes be Drug Targets? | p. 148 |
Conclusions | p. 149 |
References | p. 150 |
A Structural Analysis of Ribonuclease P | |
Introduction | p. 153 |
Chemistry of RNase P RNA | p. 155 |
Universal | p. 155 |
SN2-type Reaction | p. 155 |
pH-Dependence of the Reaction: Hydroxide Ion as the Nucleophile | p. 157 |
Metal Ions in Catalysis | p. 157 |
Phylogenetic Variation and Structure of RNase P RNA | p. 158 |
Early Studies of the RNase P RNA Structure | p. 159 |
Crystallographic Studies of Bacterial RNase P RNAs | p. 160 |
Modeling an RNase P RNA:tRNA Complex | p. 162 |
Modeling the Bacterial RNase P Holoenzyme | p. 163 |
Substrate Recognition | p. 165 |
Archaeal and Eucaryal Holoenzymes - More Proteins | p. 166 |
Concluding Remarks | p. 170 |
Acknowledgements | p. 171 |
References | p. 171 |
Group I Introns: Biochemical and Crystallographic Characterization of the Active Site Structure | |
Group I Intron Origins | p. 178 |
Group I Intron Self-splicing | p. 178 |
What has Changed in Group I Intron Knowledge in the Last Decade | p. 181 |
Structure of Group I Introns | p. 181 |
Crystallography of Group I Introns | p. 181 |
Tetrahymena LSU P4-P6 Domain | p. 182 |
Tetrahymena Intron Catalytic Core | p. 183 |
Twort orf142-I2 Ribozyme | p. 183 |
Azoarcus sp. BBH72 tRNAIle Intron | p. 184 |
Structural Basis for Group I Intron Self-splicing | p. 184 |
Recognition of the 5'-Splice Site | p. 185 |
Does the Ribozyme Undergo Conformational Changes upon PI Docking? | p. 186 |
A Binding Pocket for Guanosine | p. 187 |
Packed Stacks | p. 189 |
Biochemical Characterization of the Structure | p. 191 |
Metal Ion Binding and Specificity Switches | p. 191 |
Identification of Ligands to the Catalytic Metal Ions | p. 192 |
Correlation with Metal Ion Binding Sites within the Crystal Structures | p. 193 |
Nucleotide Analog Interference Techniques | p. 194 |
What Makes a Catalytic Site? | p. 196 |
Back to the Origins | p. 197 |
References | p. 198 |
Group II Introns: Catalysts for Splicing, Genomic Change and Evolution | |
Introduction: The Place of Group II Introns Among the Family of Ribozymes | p. 201 |
The Basic Reactions of Group II Introns | p. 201 |
The Biological Significance of Group II Introns | p. 204 |
Evolutionary Significance | p. 204 |
Significance and Prevalence in Modern Genomes | p. 204 |
The Potential Utility of Group II Introns | p. 204 |
Domains and Parts: The Anatomy of a Group II Intron | p. 205 |
Domain 1 | p. 206 |
Domain 2 | p. 206 |
Domain 3 | p. 206 |
Domain 4 | p. 206 |
Domain 5 | p. 206 |
Domain 6 | p. 207 |
Other Domains and Insertions | p. 207 |
Alternative Structural Organization and Split Introns | p. 208 |
A Big, Complicated Family: The Diversity of Group II Introns | p. 208 |
Group II Intron Tertiary Structure | p. 209 |
Group II Intron Folding Mechanisms | p. 211 |
A Slow, Direct Path to the Native State | p. 211 |
A Folding Control Element in the Center of D1 | p. 212 |
Proteins and Group II Intron Folding | p. 212 |
Setting the Stage for Catalysis: Proximity of the Splice Sites and Branch-site | p. 213 |
Recognition of Exons and Ribozyme Substrates | p. 213 |
Branch-site Recognition and the Coordination Loop | p. 213 |
A Single Active-site for Group II Intron Catalysis | p. 215 |
The Group II Intron Active-site: What are the Players? | p. 216 |
Active-site Players in D1 and Surrounding Linker Regions | p. 217 |
Domain 3 and the J2/3 Linker | p. 217 |
Domain 5: Structural and Catalytic Regions | p. 218 |
The Chemical Mechanism of Group II Intron Catalysis | p. 219 |
Proteins and Group II Intron Function | p. 221 |
Maturases | p. 221 |
CRM-domain Plant Proteins | p. 221 |
ATPase Proteins | p. 221 |
Group II Introns and Their Many Hypothetical Relatives | p. 222 |
Group II Introns: RNA Processing Enzymes, Transposons, or Tiny Living Things? | p. 223 |
References | p. 223 |
The GIR1 Branching Ribozyme | |
Introduction | p. 229 |
Distribution and Structural Organization of Twin-ribozyme Introns | p. 231 |
Biological Context | p. 234 |
Three Processing Pathways of a Twin-ribozyme Intron | p. 234 |
Processing of the I-DirI mRNA | p. 235 |
Conformational Switching in GIR1 | p. 236 |
Biochemical Characterization | p. 238 |
GIR1 Catalyzes Three Different Reactions | p. 239 |
Characterization of the Branching Reaction | p. 240 |
Biochemistry of GIR1 | p. 240 |
Modelling the Structure of GIR1 | p. 241 |
Overall Structure | p. 242 |
Coaxially Stacked Helices | p. 242 |
Junctions and Tertiary Interactions Involving Peripheral Elements | p. 245 |
The Active Site | p. 245 |
Phylogenetic Considerations | p. 247 |
Concluding Remarks | p. 248 |
References | p. 249 |
Is the Spliceosome a Ribozyme? | |
Introduction | p. 253 |
Similarity to Group II Self-splicing Introns | p. 253 |
Role of snRNA in the Spliceosome Active Site | p. 255 |
Conformation of the U2-U6 Complex and Parallels to Group II Intron Structures | p. 260 |
RNA-mediated Regulation in the Spliceosome | p. 262 |
References | p. 266 |
Peptidyl Transferase Mechanism: The Ribosome as a Ribozyme | |
Introduction: Historical Background | p. 270 |
The Ribosome | p. 271 |
Peptidyl Transfer Reaction | p. 272 |
Characteristics of the Reaction off the Ribosome | p. 273 |
Enzymology of the Peptidyl Transfer Reaction | p. 274 |
Potential Mechanisms of Rate Acceleration by the Ribosome | p. 274 |
Experimental Approaches to Reaction on the Ribosome | p. 275 |
pH-Rate Profiles | p. 277 |
Activation Parameters | p. 278 |
The Active Site | p. 279 |
Structures of the Reaction Intermediates | p. 281 |
Conformational Rearrangements of the Active Site | p. 282 |
Induced Fit | p. 282 |
Role of the P-site Substrate | p. 283 |
Conformational Flexibility of the Active Site | p. 284 |
Probing the Catalytic Mechanism: Effects of Base Substitutions | p. 285 |
Importance of the 2'-OH of A76 of the P-site tRNA | p. 286 |
Conclusions and Evolutionary Considerations | p. 287 |
References | p. 288 |
Folding Mechanisms of Group I Ribozymes | |
Introduction | p. 295 |
Multi-domain Architecture of Group I Ribozymes | p. 296 |
RNA Folding Problem | p. 297 |
Hierarchical Folding of tRNA | p. 297 |
Coupling of Secondary and Tertiary Structure | p. 298 |
Late Events: Formation of Tertiary Domains in the Tetrahymena Ribozyme | p. 298 |
Time-resolved Footprinting of Intermediates | p. 298 |
Misfolding of the Intron Core | p. 300 |
Peripheral Stability Elements | p. 300 |
Kinetic Partitioning among Parallel Folding Pathways | p. 301 |
Theory and Experiment | p. 301 |
Single Molecule Folding Studies | p. 301 |
Estimating the Flux through Footprinting Intermediates | p. 302 |
Kinetic Partitioning In Vivo | p. 302 |
Early Events: Counterion-dependent RNA Collapse | p. 302 |
Compact Non-native Form of bI5 Ribozyme | p. 303 |
Small Angle X-ray Scattering of Tetrahymena Ribozyme | p. 303 |
Native-like Folding Intermediates in the Azoarcus Ribozyme | p. 304 |
Early Folding Intermediates of the P4-P6 RNA | p. 305 |
Counterions and Folding of Group I Ribozymes | p. 305 |
Metal Ions and RNA Folding | p. 305 |
Valence and Size of Counterions Matter | p. 306 |
Specific Metal Ion Coordination and Folding | p. 307 |
Protein-dependent Folding of Group I Ribozymes | p. 307 |
Stabilization of RNA Tertiary Structure | p. 308 |
Stimulation of Refolding by RNA Chaperones | p. 308 |
Conclusion | p. 309 |
References | p. 309 |
Subject Index | p. 315 |
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