Additional roles of a peripheral loop-loop interaction in the Neurospora VS ribozyme

Area of Science:

• Biochemistry
• Molecular Biology
• RNA Biology

Background:

• RNA tertiary interactions are crucial for 3D structure and function.
• The VS ribozyme utilizes a loop-loop kissing interaction for catalytic activity.

Purpose of the Study:

• To investigate the additional roles of the VS ribozyme's kissing interaction.
• To quantify the kissing interaction's contribution to cleavage and ligation rates and equilibrium.

Main Methods:

• Utilized mutants with constitutive catalytic conformations.
• Employed mutations to disrupt or restore the kissing interaction.
• Assessed cleavage and ligation rates and equilibrium under varying Mg(2+) concentrations.

Main Results:

• The kissing interaction enhances cleavage and ligation rates by approximately 1000-fold.
• Magnesium ion (Mg(2+)) concentration significantly influences the cleavage-ligation equilibrium.
• Cleavage is favored at micromolar Mg(2+) with the kissing interaction, while ligation is favored at millimolar Mg(2+).

Conclusions:

• The kissing interaction is essential for significant rate enhancement in the VS ribozyme.
• The kissing interaction modulates the cleavage-ligation equilibrium in a Mg(2+)-dependent manner.
• This interaction plays a critical role in the catalytic mechanism and substrate processing of the VS ribozyme.