Kinetic Mechanism of the Hairpin Ribozyme

TL;DRAbstract

To investigate the relationship between RNA folding and ribozyme catalysis, we have carried out a detailed kinetic analysis of four structural derivatives of the hairpin ribozyme. Optimal and suboptimal (wild-type) substrate sequences were studied in conjunction with stabilization of helix 4, which supports formation of the catalytic core. Pre-steady-state and steady-state kinetic studies strongly support a model in which each of the ribozyme variants partitions between two major conformations leading to active and inactive ribozyme· substrate complexes. Reaction rates for cleavage, ligation, and substrate binding to both ribozyme conformations were determined. Ligation rates (3 min−1) were typically 15-fold greater than cleavage rates (0.2 min−1), demonstrating that the hairpin ribozyme is an efficient RNA ligase. On the other hand, substrate binding is very rapid (kon = 4 × 108m−1 min−1), and the ribozyme· substrate complex is very stable (KD< 25 pm; koff < 0.01 min−1). Stabilization

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