Monovalent and divalent promoted GAAA tetraloop-receptor tertiary interactions from freely diffusing single-molecule...

Area of Science:

• Biochemistry
• Molecular Biology
• Structural Biology

Background:

• RNA tertiary structure formation is essential for catalytic activity.
• Understanding specific tertiary binding interactions, like the GAAA tetraloop-receptor, is key to RNA folding.
• Previous studies often used immobilized constructs, potentially introducing artifacts.

Purpose of the Study:

• To investigate the GAAA tetraloop-receptor tertiary interaction using single-molecule fluorescence.
• To explore RNA conformational dynamics as a function of Mg(2+) and Na(+) concentrations.
• To determine if kinetic heterogeneity in docking rates is due to surface immobilization.

Main Methods:

• Single-molecule fluorescence resonance energy transfer (smFRET) studies.
• Analysis of freely diffusing RNA constructs.
• Variable concentrations of Mg(2+) and Na(+) were used to probe ion effects.

Main Results:

• Both Mg(2+) and Na(+) promote RNA docking, but with different affinities.
• Increasing Na(+) concentration leads to electrostatic relaxation and increased flexibility in undocked RNA.
• A consistent fraction (approx. 33-34%) of RNA remained undocked even at saturating Mg(2+), independent of immobilization.
• Mg(2+) and Na(+) exhibit synergistic effects on RNA folding, with lower K(D) and Hill coefficients at higher [Na(+)].

Conclusions:

• Freely diffusing single-molecule studies accurately reflect RNA folding dynamics.
• The GAAA tetraloop-receptor interaction is modulated by both Mg(2+) and Na(+) in a synergistic manner.
• Kinetic heterogeneity in RNA docking is an intrinsic property, not an artifact of immobilization.