2013 Rustbelt RNA Meeting
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