A study of the metal concentration and metal identity effects on structural dynamics of the non-cleavable hammerhead ribozyme by single-molecule fluorescence resonance energy transfer

There have been numerous studies to reveal the correlation between structural dynamics and function of ribozymes. To better understand how structure affects the function of ribozyme, I first investigated the Mg2+-dependent structural dynamics of the non-cleavable hammerhead ribozyme (HHRz) by single-molecule FRET (sm-FRET). From the sm-FRET measurements performed, I have observed the Mg2+-dependent folding dynamics of the HHRz, accompanied by an increase (decrease) in the rate constant for docking (undocking) as a function of the Mg2+ concentration. More interestingly, the observed kinetic and thermodynamic heterogeneous behaviors in the single molecule FRET trace at all tested Mg2+ concentrations indicate the presence of the conformational isomers in multiple equilibria. Also, I have investigated the structural dynamics of the non-cleavable HHRz in the presence of various (mono/di/trivalent) metal cations at the single molecule level to see whether identity of metal ion affects the docked population of HHRz differently. The results show little metal identity effects, indicating that non-specific interaction of metal cations to the ribozyme is more important in promoting docked conformation of the ribozyme. Also, the complex undocking kinetics with a strong stochastic preference between the subsequent docked dwell times was found to mainly determine the equilibrium of structural dynamics of the HHRz for all tested metal ions. Thus, the single-molecule FRET study revealed complex thermodynamic and kinetic behaviors of the HHRz docking, independent on the identity of metal ions, which could not be observed previously in ensemble FRET studies.