KINETIC MECHANISM OF THE HAIRPIN RIBOZYME - IDENTIFICATION AND CHARACTERIZATION OF 2 NONEXCHANGEABLE CONFORMATIONS

To investigate the relationship between RNA folding and ribozyme catal ysis, we have carried out a detailed kinetic analysis of four structur al derivatives of the hairpin ribozyme. Optimal and suboptimal (wild-t ype) substrate sequences were studied in conjunction with stabilizatio n of helix 4, which supports formation of the catalytic core. Pre-stea dy-state and steady-state kinetic studies strongly support a model in which each of the ribozyme variants partitions between two major confo rmations leading to active and inactive ribozyme substrate complexes. Reaction rates for cleavage, ligation, and substrate binding to both r ibozyme conformations were determined. Ligation rates (3, min(-1)) wer e typically 15-fold greater than cleavage rates (0.2 min(-1)), demonst rating that the hairpin ribozyme is an efficient RNA ligase. On the ot her hand, substrate binding is very rapid (k(on) = 4 x 10(8) M-1 min(- 1)), and the ribozyme substrate complex is very stable (K-D < 25 pm; k (off) < 0.01 min(-1)). Stabilization of helix 4 increases the proporti on of RNA molecules folded into the active conformation, and enhances substrate association and ligation rates. These effects can be explain ed by stabilization of the catalytic core of the ribozyme, Rigorous co nsideration of conformational isomers and their intrinsic kinetic prop erties was necessary for development of a kinetic scheme for the riboz yme-catalyzed reaction.