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Cryoenzymology of the hammerhead ribozyme

Published online by Cambridge University Press:  01 October 1998

ANDREW L. FEIG ,
GLENYSS E. AMMONS  and
OLKE C. UHLENBECK
ANDREW L. FEIG
Affiliation:
Department of Chemistry and Biochemistry, University of Colorado, Boulder, Colorado 80309, USA
GLENYSS E. AMMONS
Affiliation:
Department of Chemistry and Biochemistry, University of Colorado, Boulder, Colorado 80309, USA
OLKE C. UHLENBECK
Affiliation:
Department of Chemistry and Biochemistry, University of Colorado, Boulder, Colorado 80309, USA
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Abstract

The technique of cryoenzymology has been applied to the hammerhead ribozyme in an attempt to uncover a structural rearrangement step prior to cleavage. Several cryosolvents were tested and 40% (v/v) methanol in water was found to perturb the system only minimally. This solvent allowed the measurement of ribozyme activity between 30 and −33°C. Eyring plots are linear down to −27°C, but a drastic reduction in activity occurs below this temperature. However, even at extremely low temperatures, the rate is still quite pH dependent, suggesting that the chemical step rather than a structural rearrangement is still rate-limiting. The nonlinearity of the Eyring plot may be the result of a transition to a cold-denatured state or a glassed state.

Keywords

conformational changekineticstemperature dependence
Type
Research Article
Information
RNA , Volume 4 , Issue 10 , October 1998 , pp. 1251 - 1258
Copyright
© 1998 RNA Society

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