A kinetic and thermodynamic framework for the hammerhead ribozyme reaction.
A hammerhead ribozyme (HH16) with eight potential base pairs in each of the substrate recognition helices stabilized product binding sufficiently to enable investigation of the ligation of oligonucleotides bound to the ribozyme. All individual rate constants for product association and dissociation were determined. The following conclusions were obtained for HH16 from the analysis performed at 50 mM Tris, pH 7.5, 10 mM MgCl2, and 25 degrees C. (1) HH16 cleaves bound substrate with a rate constant of k2 = 1 min-1, similar to rate constants obtained with other hammerhead ribozymes. (2) k-2, the rate of ligation of the 5' product and 3' product to form substrate, equaled 0.008 min-1, indicating an approximately 100-fold preference for the formation of products on the ribozyme. This internal equilibrium, compared with that for the overall solution reaction, gives an effective concentration (EC) of 10(-2) M for the two products bound to the ribozyme. This low EC suggests that upon cleavage of S the hammerhead complex acquires a "floppiness" which provides an entropic advantage for the formation of products on the ribozyme. (3) Product and substrate association rate constants were in the range of 10(7)-10(8) M-1 min-1, comparable to values determined for short helices. (4) The stabilities of ribozyme/product complexes were similar to affinities predicted from helix-coil transitions of simple RNA duplexes, providing no indication of additional tertiary interactions. The products, P1 and P2, stabilize one another 4-fold on the ribozyme. (5) The dissociation constant for the binding of the substrate to the ribozyme was estimated to be about 10(-17) M. These results allowed the construction of a free energy profile for the reaction of HH16, and provide a basis for future mechanistic studies.
Hertel
K J
KJ
Department of Chemistry and Biochemistry, University of Colorado, Boulder 80309-0215.
Herschlag
D
D
Uhlenbeck
O C
OC
D001482:Base Composition; D001483:Base Sequence; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D009841:Oligonucleotides / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D016337:RNA, Catalytic / Q000737:chemistry* / Q000378:metabolism*; D013816:Thermodynamics; D014764:Viral Proteins
D009841:Oligonucleotides; D016337:RNA, Catalytic; D014764:Viral Proteins; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases