Ribozymes and RNA Catalysis

Preface: Foreword: Twenty-five years of ribozymes;

Chapter 1: Ribozymes and RNA Catalysis Introduction and Primer;

1.1. What are ribozymes?;

1.2. What is the role of ribozymes in cells?;

1.3. Ribozymes bring about significant rate enhancements;

1.4. Why study ribozymes?;

1.5. Folding RNA into the active conformation;

1.6. The catalytic resources of RNA - making a lot of a little;

1.7. Mechanisms and catalytic strategies of ribozymes;

1.8. The impact of new methodologies to study ribozymes;

1.9. Finally;

Chapter 2: Proton Transfer in Ribozyme Catalysis;

2.1 Scope of Chapter and Rationale;

2.2 Overview of Proton Transfer Chemistry;

2.3 General Considerations for Proton Transfer in RNA Enzymes;

2.3.1 Classes of Protonation Sites in RNA;

2.3.2 Driving Forces for pKa Shifting in RNA;

2.3.3 Quantitative Contributions of Proton Transfer to RNA Catalysis;

2.4 Proton Transfer in Small Ribozymes: 5 case studies;

2.4.1 Why Small Ribozymes?;

2.4.2 Proton Transfer in the Hepatitis Delta Virus Ribozyme;

2.4.3 Proton Transfer in the Hairpin Ribozyme;

2.4.4 Proton Transfer in the Hammerhead Ribozyme;

2.4.5 Proton Transfer in the VS Ribozyme;

2.4.6 Proton Transfer in the glmS Ribozyme;

2.5 Conclusion and Perspectives;

2.6 References and Footnotes;

Chapter 3: Finding the hammerhead active site;

3.1. Introduction;

3.2. Background;

3.3. Experimental data;

3.1.1 Mechanistic Hypothesis Leads to Identification and Functional Test of Active Site Components;

3.1.2. Structural Hypothesis-Large-scale Conformational Changes are Required for Catalysis;

3.1.3. Molecular Modeling of a Hammerhead Active Fold that Satisfies Structural and Biochemical Constraints;

3.4. Current Status and Future Prospects;

Chapter 4: Hammerhead Ribozyme Crystal Structures and Catalysis;

4.1 Introduction;

4.2 A Catalytic RNA Prototype;

4.3 A Small Ribozyme;

4.4 The Chemistry of Phosphodiester Bond Isomerization;

4.5 The Hammerhead Ribozyme Structure Nailed Down;

4.6 Catalysis in the Crystal;

4.7 Making Movies From Crystallographic Snapshots;

4.8 An Ever-Growing List of Concerns;

4.9 Occam's Razor Can Slit Your Throat;

4.10 The Structure of a Full-Length Hammerhead Ribozyme;

4.11 Do the Minimal and Full-Length Hammerhead Crystal Structures Have Anything in Common?;

4.12 How the Does the Minimal Hammerhead Work?;

4.13 A Movie Sequel with a Happy Ending;

4.14 Concluding remarks;

Chapter 5: The Hairpin and Varkud Satellite Ribozymes;

5.1. The nucleolytic ribozymes;

5.2. The hairpin ribozyme;

5.2.1 The structure of the hairpin ribozyme;

5.2.2 Metal ion-dependent folding of the hairpin ribozyme;

5.2.3 Observing the cleavage and ligation activities of the hairpin ribozyme;

5.2.4 The mechanism of the hairpin ribozyme;

5.3. The VS ribozyme;

5.3.1 The structure of the VS ribozyme;

5.3.2 The structure of the substrate;

5.3.3 The location of the substrate;

5.3.4 The active site of the VS ribozyme;

5.3.5 Candidate catalytic nucleobases;

5.3.6 The mechanism of the VS ribozyme;

5.4. Some striking similarities between the hairpin and VS ribozymes;

Chapter 6: Catalytic Mechanism of the HDV Ribozyme;

6.1. Introduction;

6.1.1. Hepatitis Delta Virus Biology;

6.1.2. Cleavage Reactions of Small Ribozymes;

6.2. HDV structure;

6.2.1. Determination of Crystal Structures;

6.2.2. Structure Overview;

6.2.3. Active Site;

6.3. Catalytic Strategies for RNA Cleavage;

6.4. The Active Site Nucleobase: C75;

6.4.1. Exogenous Base Rescue Reactions;

6.4.2. The Role of C75 in HDV Catalysis;

6.4.3. Resolving the Kinetic Ambiguity;

6.5. Metal ions in the HDV Ribozyme;

6.5.1. Structural Metal Ions;

6.5.2. Catalytic Metal Ions;

6.6. Contributions of Non-active-site Structures to Catalysis;

6.7. Dynamics in HDV Function;

6.8. Varieties of Experimental Systems;

6.9. Models for HDV Catalysis;

6.10. Conclusion;

Chapter 7: Mammalian self-cleaving ribozymes;

7.1 Introduction;

7.2 General features of small self-cleaving sequences;

7.3 Genome-wide selection of self-cleaving ribozymes;

7.4 The CPEB3 ribozyme;

7.4.1 Expression of the CPEB3 ribozyme;

7.4.2 Structural features of the CPEB3 and HDV ribozymes;

7.4.3 Linkage of HDV to the human transcriptome;

7.5 Possible biological roles of self-cleaving ribozymes;

7.6 Closing remarks;

Chapter 8: The Structure and Action of glmS Ribozymes;

8.1 Introduction;

8.2 Biochemical characteristics of glmS ribozymes;

8.2.1 Divalent metal ions support structure and not chemistry;

8.2.2 Ligand specificity of glmS ribozymes;

8.2.3 Evidence for a coenzyme role for GlcN6P;

8.3 Atomic-resolution structure of glmS ribozymes;

8.3.1 Secondary and tertiary structures of glmS ribozymes;

8.3.2 Metabolite recognition by glmS ribozymes;

8.4 Mechanism of glmS ribozyme self-cleavage;

8.5 Can glmS ribozymes be drug targets?;

8.6 Conclusions;

Chapter 9: A Structural Analysis of Ribonuclease P. 9.1 Introduction;

9.2 Chemistry of RNase P RNA;

9.2.1 Universal;

9.2.2 SN2-type reaction;

9.2.3 pH-dependence of the reaction: hydroxide ion as the nucleophile;

9.2.4 Metal ions in catalysis ;

9.3 Phylogenetic variation and structure of RNase P RNA;

9.4 Early studies of the RNase P RNA structure;

9.5 Crystallographic studies of bacterial RNase P RNAs;

9.6 Modeling an RNase P RNA:tRNA complex;

9.7 Modeling the bacterial RNase P holoenzyme;

9.8 Substrate recognition;

9.9 Archaeal and Eucaryal holoenzymes - more proteins;

9.10 Concluding remarks;

Chapter 10: Group I Introns: Biochemical and Crystallographic Characterization of the Active Site Structure;

10.1. Group I intron origins;

10.2 Group I intron self-splicing;

10.3 What has changed in group I intron knowledge in the last decade;

10.4 The structure of group I introns;

10.5 Crystallography of group I introns;

10.5.1 The Tetrahymena LSU P4-P6 domain;

10.5.2 The Tetrahymena intron catalytic core;

10.5.3 The Twort orf142-I2 ribozyme;

10.5.4 The Azoarcus sp. BBH72 tRNAile intron;

10.6 The structural basis for group I intron self-splicing;

10.6.1 Recognition of the 5' splice site;

10.6.2 Does the ribozyme undergo conformational changes upon P1 docking?;

10.6.3 A binding pocket for guanosine;

10.6.4 Packed stacks;

10.7 Biochemical characterization of the structure;

10.7.1 Metal ion binding and specificity switches;

10.7.2 Identification of ligands to the catalytic metal ions;

10.7.3 Correlation with metal ion binding sites within the crystal structures;

10.7.4 Nucleotide analog interference techniques;

10.8 What makes a catalytic site?;

10.9 Back to the origins;

Chapter 10: Group II introns: catalysts for splicing, genomic change and evolution;

11.1 Introduction: The place of group II introns among the family of ribozymes;

11.2 The basic reactions of group II introns;

11.3 The biological significance of group II introns;

11.3.1 Evolutionary significance;

11.3.2 Significance and prevalence in modern genomes;

11.4 Domains and parts: the anatomy of a group II intron;

11.4.1. Domain 1;

11.4.2. Domain 2;

11.4.3. Domain 3;

11.4.4. Domain 4;

11.4.5. Domain 5;

11.4.6. Domain 6;

11.4.7. Other domains and insertions;

11.4.8. Alternative structural organization and split introns;

11.5. A big, complicated family: the diversity of group II introns;

11.6. Group II intron tertiary structure;

11.7. Group II intron folding mechanisms;

11.7.1. A slow, direct path to the native state:;

11.7.2. A folding control element in the center of D1;

11.7.3. Proteins and group II intron folding;

11.8 Setting the stage for catalysis: proximity of the splice sites and branch-site;

11.8.1. Recognition of exons and ribozyme substrates;

11.8.2. Branch-site recognition and the coordination loop;

11.9. A single active-site for group II intron catalysis;

11.10 The group II intron active-site: what are the players?;

11.10.1. Active-site players in D1 and surrounding linker regions;

11.10.2. Domain 3 and the J2/3 linker;

11.10.3. Domain 5: structural and catalytic regions;

11.11 The chemical mechanism of group II intron catalysis;

11.12 Proteins and group II intron function;

11.12.1. Maturases;

11.12.2. CRM-domain plant proteins;

11.12.3. ATPase proteins;

11.13 Group II introns and their many hypothetical relatives;

11.14 Group II introns: RNA processing enzymes, transposons, or tiny living things?;

Chapter 12: The GIR1 branching ribozyme;

12.1 Introduction;

12.2 Distribution and structural organization of twin-ribozyme introns;

12.3 The biological context;

12.3.1 Three processing pathways of a twin-ribozyme intron;

12.3.2 Processing of the I-DirI mRNA;

12.3.3 Conformational switching in GIR1;

12.4 Biochemical characterization;

12.4.1 GIR1 catalyzes three different reactions;

12.4.2 Characterization of the branching reaction;

12.4.3 The biochemistry of GIR1;

12.5 Modelling the structure of GIR1;

12.5.1 The overall structure;

12.5.2 Coaxially stacked helices;

12.5.3 The junctions and tertiary interactions involving peripheral elements;

12.5.4 The active site;

12.6 Phylogenetic considerations;

12.7 Concluding remarks;

Chapter 13: Is the spliceosome a ribozyme?;

13.1 Similarity to group II self-splicing introns;

13.2 Role of snRNA in the spliceosome active site;

13.3 Conformation of the U2-U6 complex and parallels to group II intron structures;

13.4 RNA-mediated regulation in the spliceosome;

Chapter 14: Peptidyl Transferase Mechanism: The Ribosome as a Ribozyme;

14.1 Introduction: Historical background;

14.2 The ribosome;

14.3 The peptidyl transfer reaction;

14.3.1 Characteristics of the reaction off the ribosome;

14.3.2 Enzymology of the peptidyl transfer reaction;

14.3.2.1 Potential mechanisms of rate acceleration by the ribosome;

14.3.2.2 Experimental approaches to reaction on the ribosome;

14.3.2.3 pH-rate profiles;

14.3.2.4 Activation parameters;

14.4 The active site;

14.4.1 Structures of the reaction intermediates;

14.4.2 Conformational rearragements of the active site;

14.4.2.1 Induced fit;

14.4.2.2 Role of the P-site substrate;

14.4.2.3 Conformational flexibility of the active site;

14.4.3 Probing the catalytic mechanism: Effects of base substitutions;

14.4.4. Importance of the 2?-OH of A76 of the P-site tRNA;

14.5 Conclusions and evolutionary considerations;

Chapter 15: Folding Mechanisms of Group I Ribozymes;

15.1. The multi-domain architecture of group I ribozymes;

15.2. RNA Folding Problem;

15.2.1 Hierarchical Folding of tRNA;

15.2.2 Coupling of Secondary and Tertiary Structure;

15.3. Late events: Formation of Tertiary Domains in the Tetrahymena Ribozyme;

15.3.1 Time-resolved Footprinting of Intermediates;

15.3.2 Misfolding of the intron core;

15.3.3 Peripheral Stability Elements;

15.4. Kinetic Partitioning among Parallel Folding Pathways;

15.4.1 Theory and Experiment;

15.4.2 Single Molecule Folding Studies;

15.4.3 Estimating the Flux through Footprinting Intermediates;

15.4.4. Kinetic Partitioning in vivo;

15.5. Early Events: Counterion-dependent RNA collapse;

15.5.1 Compact Non-Native Form of bI5 Ribozyme;

15.5.2 Small Angle X-ray Scattering of Tetrahymena Ribozyme;

15.5.3 Native-like Folding Intermediates in the Azoarcus Ribozyme;

15.5.4 Early Folding Intermediates of the P4-P6 RNA;

15.6. Counterions and Folding of Group I Ribozymes;

15.6.1 Metal Ions and RNA Folding;

15.6.2 Valence and Size of Counterions Matter;

15.6.3 Specific Metal Ion Coordination and Folding;

15.7. Protein-dependent folding of group I ribozymes;

15.7.1 Stabilization of RNA Tertiary Structure;

15.7.2 Stimulation of Refolding by RNA Chaperones;

15.8. Conclusion;