Biophysical and biochemical investigations of RNA catalysis in the hammerhead ribozyme

Abstract

1. How do ribozymes work? 241

2. The hammerhead RNA as a prototype ribozyme 242

2.1 RNA enzymes 242

2.2 Satellite self-cleaving RNAs 242

2.3 Hammerhead RNAs and hammerhead ribozymes 244

3. The chemical mechanism of hammerhead RNA self-cleavage 246

3.1 Phosphodiester isomerization via an S_N2(P) reaction 247

3.2 The canonical role of divalent metal ions in the hammerhead ribozyme reaction 251

3.3 The hammerhead ribozyme does not actually require metal ions for catalysis 254

3.4 Hammerhead RNA enzyme kinetics 257

4. Sequence requirements for hammerhead RNA self-cleavage 260

4.1 The conserved core, mutagenesis and functional group modifications 260

4.2 Ground-state vs. transition-state effects 261

5. The three-dimensional structure of the hammerhead ribozyme 262

5.1 Enzyme–inhibitor complexes 262

5.2 Enzyme–substrate complex in the initial state 264

5.3 Hammerhead ribozyme self-cleavage in the crystal 264

5.4 The requirement for a conformational change 265

5.5 Capture of conformational intermediates using crystallographic freeze-trapping 266

5.6 The structure of a hammerhead ribozyme ‘early’ conformational intermediate 267

5.7 The structure of a hammerhead ribozyme ‘later’ conformational intermediate 268

5.8 Is the conformational change pH dependent? 269

5.9 Isolating the structure of the cleavage product 271

5.10 Evidence for and against additional large-scale conformation changes 274

5.11 NMR spectroscopic studies of the hammerhead ribozyme 278

6. Concluding remarks 280

7. Acknowledgements 281

8. References 281

1. How do ribozymes work? 241

The discovery that RNA can be an enzyme (Guerrier-Takada et al. 1983; Zaug & Cech, 1986) has created the fundamental question of how RNA enzymes work. Before this discovery, it was generally assumed that proteins were the only biopolymers that had sufficient complexity and chemical heterogeneity to catalyze biochemical reactions. Clearly, RNA can adopt sufficiently complex tertiary structures that make catalysis possible. How does the three- dimensional structure of an RNA endow it with catalytic activity? What structural and functional principles are unique to RNA enzymes (or ribozymes), and what principles are so fundamental that they are shared with protein enzymes?