Kinetic mechanism of the hairpin ribozyme. Identification and characterization of two nonexchangeable conformations.
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 x 10(8) M-1 min-1), and the ribozyme. substrate complex is very stable (KD < 25 pM; koff < 0.01 min-1). Stabilization of helix 4 increases the proportion of RNA molecules folded into the active conformation, and enhances substrate association and ligation rates. These effects can be explained by stabilization of the catalytic core of the ribozyme. Rigorous consideration of conformational isomers and their intrinsic kinetic properties was necessary for development of a kinetic scheme for the ribozyme-catalyzed reaction.
Esteban
J A
JA
Markey Center for Molecular Genetics, Department of Microbiology and Molecular Genetics, The University of Vermont, Burlington, Vermont 05405, USA.
Banerjee
A R
AR
Burke
J M
JM
D001483:Base Sequence; D002384:Catalysis; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D015195:Drug Design; D007700:Kinetics; D008956:Models, Chemical; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D012313:RNA / Q000737:chemistry / Q000378:metabolism; D016337:RNA, Catalytic / Q000737:chemistry* / Q000378:metabolism; D013698:Templates, Genetic
D016337:RNA, Catalytic; D012313:RNA; D004247:DNA