TERTIARY STRUCTURE FORMATION IN THE HAIRPIN RIBOZYME MONITORED BY FLUORESCENCE RESONANCE ENERGY-TRANSFER

The complex formed by the hairpin ribozyme and its substrate consists of two independently folding domains which interact to form a catalyti c structure. Fluorescence resonance energy transfer methods permit us to study reversible transitions or the complex between open and closed forms. Results indicate that docking of the domains is required for b oth the cleavage and ligation reactions. Docking is rate-limiting for ligation (2 min(-1)) but not for cleavage, where docking (0.5 min(-1)) precedes a rate-limiting conformational transition or slow-reaction c hemistry. Strikingly, most modifications to the RNA (such as a G(+1)A mutation in the substrate) or reaction conditions (such as omission of divalent metal ion cofactors) which inhibit catalysis do so by preven ting docking. This demonstrates directly that mutations and modificati ons which inhibit a step following substrate binding are not necessari ly involved in catalysis. An improved kinetic description of the catal ytic cycle is derived, including specific structural transitions.