2013 Rustbelt RNA Meeting







Talk abstracts

Talk on Saturday 09:15-09:30am submitted by Neil White

Kinetics and Thermodynamics of the Juctionless Hairpin Ribozyme

Neil A. White (Biochemistry and Molecular Biology, Michigan State University), Charles G. Hoogstraten (Biochemistry and Molecular Biology, Michigan State University)

The hairpin ribozyme is a catalytic RNA that performs self-cleavage. It consists of two loops, denoted A and B. The two loops undergo major structural rearrangements to form an intricate RNA-RNA tertiary interface. In nature the two loops are connected by a four-way junction. As a complement to spectroscopic studies, we are examining the docking properties of a junctionless (trans-docking) system in which the two loops reside on separate molecules. By utilizing temperature-dependent surface plasmon resonance we have been able to characterize the thermodynamics and kinetics of docking for the juctionless hairpin ribozyme. Using mutations at the acid-base catalytic residue A38, we have also investigated the effect of the 2’-O-methyl modification at the active site that is commonly used to prevent cleavage in biophysical and structural studies. In agreement with recent single-molecule kinetic fingerprinting studies, we find a significant increase in binding affinity in the presence of the native 2’-OH rather than the 2’-O-methyl modification at the active site. Interestingly, docking of the 2’-OH species is more enthalpically favorable but less entropically favorable than the 2’-O-methyl species. Activation energies were determined for docking and undocking in the 2’-OH species but were undeterminable in the 2’-O-methyl species due to nonlinear Arrhenius curves, suggesting a significant disruption of the energy landscape among the two species. Comparison with results in other systems emphasizes the diversity of thermodynamic contributions for RNA-RNA tertiary structure formation.

Keywords: hairpin ribozyme, thermodynamics, RNA-RNA tertiary structure