Circular substrates of the hammerhead ribozyme shift the internal equilibrium further toward cleavage.
To test whether the Y-shaped conformation of the hammerhead ribozyme is maintained throughout the catalytic pathway, the cleavage properties of circular substrates which bind the ribozyme through helices I and II were determined. Constraining the position of helices I and II in this manner did not significantly alter the rate constant for cleavage, consistent with no large rearrangement of the helices occurring during catalysis. Unexpectedly, the "internal" equilibrium between the cleavage and ligation reactions for the circular hammerheads was shifted further toward cleavage. This effect was due to the rate of ligation of the circular substrate being slower than the corresponding linear substrate. The temperature dependence of the internal equilibrium of the circular substrate revealed that although restricting the flexibility of the hammerhead reduced the favorable entropy change associated with cleavage as expected, the unfavorable enthalpy change was reduced as well, resulting in greater overall cleavage.
Stage-Zimmermann
T K
TK
Department of Chemistry and Biochemistry, University of Colorado, Boulder 80309-0215, USA.
Uhlenbeck
O C
OC
D001483:Base Sequence; D004270:DNA, Circular / Q000737:chemistry / Q000378:metabolism*; D006868:Hydrolysis; D007700:Kinetics; D015636:Magnesium Chloride / Q000378:metabolism; D008969:Molecular Sequence Data; D016337:RNA, Catalytic / Q000737:chemistry / Q000378:metabolism*; D013379:Substrate Specificity; D013816:Thermodynamics
D004270:DNA, Circular; D016337:RNA, Catalytic; D015636:Magnesium Chloride