Current Status and Future Prospects p. 46 Acknowledgements p. 46 References p. 46 Hammerhead Ribozyme Crystal Structures and Catalysis

Transcription

1 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 pk 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 Structural Hypothesis - Large-scale Conformational Changes are Required for Catalysis Molecular Modeling of a Hammerhead Active Fold that Satisfies Structural and Biochemical Constraints p. 40 p. 41 p. 43 Current Status and Future Prospects p. 46 Acknowledgements p. 46 References p. 46 Hammerhead Ribozyme Crystal Structures and Catalysis

2 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

3 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

4 A Structural Analysis of Ribonuclease P Introduction p. 153 Chemistry of RNase P RNA p. 155 Universal p. 155 S 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 trna 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

5 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

6 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

7 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 Table of Contents provided by Blackwell's Book Services and R.R. Bowker. Used with permission.