An RNA internal loop acts as a hinge to facilitate ribozyme folding and catalysis

Area of Science:

• Molecular Biology
• RNA Structure and Dynamics
• Biochemistry

Background:

• RNA molecules feature helical regions and internal loops, some of which form stable structures.
• The P4-P6 domain of the Tetrahymena self-splicing group I intron contains a critical J5/5a internal loop.

Purpose of the Study:

• To investigate the function and dynamics of the J5/5a internal loop in the Tetrahymena group I intron P4-P6 domain.
• To understand the role of this loop in RNA folding and tertiary contact formation.

Main Methods:

• Site-directed mutagenesis of the J5/5a loop region.
• Nondenaturing gel electrophoresis to assess RNA folding and mobility.
• Fe(II)-EDTA cleavage assays to probe RNA structure.
• Analysis of reconstituted introns with mutant P4-P6 RNAs.

Main Results:

• Mutations in the J5/5a loop affected P4-P6 RNA folding and stability, with less structured loops behaving more like wild-type.
• Magnesium ion (Mg2+) concentration required for folding rescue correlated with loop structure.
• Activity of reconstituted introns correlated with J5/5a loop mutations.
• P4-P6 RNA exists in a two-state equilibrium between folded and unfolded states in solution.

Conclusions:

• The J5/5a internal loop functions primarily as a flexible hinge, enabling tertiary interactions between helical regions.
• Specific bases in the loop contribute to folding to a lesser extent.
• Maintaining an unstructured state of the J5/5a loop in the unfolded state is essential.
• RNA structure determination requires integration of mutational and thermodynamic analyses alongside structural data.