Tertiary interactions determine the accuracy of RNA folding

Area of Science:

• Molecular Biology
• Biochemistry
• Structural Biology

Background:

• RNA molecules fold into specific three-dimensional structures essential for their cellular functions.
• The precise mechanisms by which RNA achieves its native structure remain incompletely understood.

Purpose of the Study:

• To investigate the role of tertiary structure stability in the speed and accuracy of RNA folding.
• To understand how disruptions in specific RNA interactions affect folding pathways.

Main Methods:

• Site-directed mutagenesis was used to perturb the tetraloop-receptor interaction in a bacterial group I ribozyme.
• Nondenaturing polyacrylamide gel electrophoresis (PAGE) and RNase T1 digestion were employed to analyze base pairing homogeneity.
• Time-resolved hydroxyl radical footprinting was utilized to monitor folding kinetics.

Main Results:

• Destabilizing tertiary interactions by 2-3 kcal/mol led to less homogeneous base pair formation during folding.
• Wild-type ribozyme folding completed within 5-20 ms.
• Mutant ribozymes showed significantly slower folding, with a portion requiring seconds to fold via nonnative intermediates.

Conclusions:

• Stable tertiary interactions cooperatively stabilize the native RNA structure.
• Disrupting these interactions introduces alternative, slower folding pathways, deviating from the native energy landscape.
• Enhanced stability of tertiary structure is crucial for rapid and accurate RNA folding to the native state.