Structure-function analysis from the outside in: long-range tertiary contacts in RNA exhibit distinct catalytic roles

Area of Science:

• Molecular Biology
• Structural Biology
• Biochemistry

Background:

• The Tetrahymena group I ribozyme features a catalytic core surrounded by peripheral elements.
• Five long-range tertiary contacts stabilize these peripheral elements, contributing to the ribozyme's overall structure and function.

Purpose of the Study:

• To investigate the structure-function relationships of the five long-range tertiary contacts in the Tetrahymena group I ribozyme.
• To determine the specific roles of individual tertiary contacts in ribozyme stability, catalysis, and substrate/nucleophile binding.

Main Methods:

• Site-directed mutagenesis to ablate specific tertiary contacts.
• Enzyme kinetics and thermodynamics to dissect reaction steps.
• Hydroxyl radical footprinting to assess structural changes.

Main Results:

• Mutational disruption of tertiary contacts destabilized the ribozyme and impaired catalysis.
• Specific contacts (P14, MC/MCR, L9/P5) differentially affected substrate docking and guanosine binding.
• P13 and tetraloop/tetraloop receptor (TL/TLR) mutations had minimal functional or structural impact.
• Structural changes in mutants extended to the catalytic core, indicating allosteric pathways.

Conclusions:

• Peripheral elements and their tertiary contacts are crucial for ribozyme stability and catalysis.
• Individual tertiary contacts exhibit modular functional specialization, influencing specific steps in the catalytic process.
• Distinct allosteric pathways link tertiary contacts to the ribozyme's conserved core, highlighting a fundamental strategy in RNA structure-function.