Biophysical and biochemical investigations of RNA catalysis in the hammerhead ribozyme.

Abstract

1. How do ribozymes work? 2412. The hammerhead RNA as a prototype ribozyme 2422.1 RNA enzymes 2422.2 Satellite self-cleaving RNAs 2422.3 Hammerhead RNAs and hammerhead ribozymes 2443. The chemical mechanism of hammerhead RNA self-cleavage 2463.1 Phosphodiester isomerization via an SN2(P) reaction 2473.2 The canonical role of divalent metal ions in the hammerhead ribozyme reaction 2513.3 The hammerhead ribozyme does not actually require metal ions for catalysis 2543.4 Hammerhead RNA enzyme kinetics 2574. Sequence requirements for hammerhead RNA self-cleavage 2604.1 The conserved core, mutagenesis and functional group modifications 2604.2 Ground-state vs. transition-state effects 2615. The three-dimensional structure of the hammerhead ribozyme 2625.1 Enzyme–inhibitor complexes 2625.2 Enzyme–substrate complex in the initial state 2645.3 Hammerhead ribozyme self-cleavage in the crystal 2645.4 The requirement for a conformational change 2655.5 Capture of conformational intermediates using crystallographic freeze-trapping 2665.6 The structure of a hammerhead ribozyme ‘early’ conformational intermediate 2675.7 The structure of a hammerhead ribozyme ‘later’ conformational intermediate 2685.8 Is the conformational change pH dependent? 2695.9 Isolating the structure of the cleavage product 2715.10 Evidence for and against additional large-scale conformation changes 2745.11 NMR spectroscopic studies of the hammerhead ribozyme 2786. Concluding remarks 2807. Acknowledgements 2818. References 2811. How do ribozymes work? 241The 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?