Metal ion dependence, thermodynamics, and *kinetics of the GAAA tetraloop-receptor RNA tertiary interaction

The GAAA tetraloop-receptor is a commonly occurring tertiary interaction motif in RNA. This motif is often found in combination with other tertiary interactions in complex RNA structures, making it difficult to measure directly the contribution of a single GAAA tetraloop-receptor interaction to the folding of an RNA. To investigate the thermodynamics and kinetics for an isolated GAAA tetraloop-receptor interaction, a construct was designed where the two domains are connected by a flexible single-stranded linker. Fluorescence resonance energy transfer (FRET) was used to probe the intramolecular docking of the GAAA tetraloop to the receptor.;The GAAA tetraloop-receptor interaction was characterized extensively by ensemble FRET experiments. The metal ion requirements for docking of the GAAA tetraloop-receptor were determined. The results showed that docking was induced by a variety of cations, where the charge of the ion was the most important factor in determining the concentration of the ion required to promote docking. Stopped-flow fluorescence techniques were used to measure docking kinetics and yielded the apparent activation energy for the docking reaction. The sensitivity of the docking reaction to the length and sequence of the single-stranded region connecting the tetraloop and receptor domains was also investigated by both ensemble and single-molecule FRET experiments.;The equilibrium and kinetics for docking and undocking of the GAAA tetraloop-receptor motif were directly probed by single-molecule FRET. These experiments yielded the free energy change for the docking reaction. Many RNA tertiary structures undergo dynamic motions that play a significant role in their function. The single-molecule study revealed that the GAAA tetraloop-receptor interaction is dynamic, with significant populations of docked and undocked conformations that interconvert over a 10-2-10-1 s time scale. The influence of Mg2+ on both the docking and undocking reaction rate constants was explored.;The effects of hydrostatic pressure and osmotic stress on RNA tertiary structure are not well understood. The GAAA tetraloop-receptor construct was used as a model system to test how these parameters influence the stability of an RNA tertiary interaction. The results yielded the thermodynamic parameter Delta V for the docking reaction and provided insight into the role of bound water molecules in forming the GAAA tetraloop-receptor interaction.