Operative Binding of Class I Release Factors and YaeJ Stabilizes the Ribosome in the Nonrotated State

Area of Science:

• Molecular Biology
• Structural Biology
• Biochemistry

Background:

• Ribosome function involves intersubunit rotation between rotated and nonrotated states during mRNA translation.
• Class I release factors (RFs) terminate translation by binding stop codons, while rescue factors like YaeJ handle stalled ribosomes.
• Previous structural studies indicated a nonrotated state upon factor binding but lacked dynamic insights.

Purpose of the Study:

• To investigate the dynamic effects of RF1, RF2, and YaeJ binding on ribosome intersubunit rotation.
• To determine the influence of cognate versus noncognate codon binding on ribosome conformation.
• To elucidate the role of these factors in the termination and recycling of bacterial translation.

Main Methods:

• Single-molecule Förster resonance energy transfer (smFRET) was employed to monitor ribosome dynamics.
• Wild-type and mutant forms of RF1, RF2, and YaeJ were used.
• Analysis focused on shifts in the population of rotated versus nonrotated ribosome states.

Main Results:

• RF1 and RF2 binding shifted posthydrolysis ribosomes to the nonrotated state, contingent on cognate stop codon recognition.
• YaeJ binding stabilized nonstop ribosomal complexes in the nonrotated state.
• Cognate codon binding is crucial for inducing the nonrotated conformation.

Conclusions:

• Ribosome release factors and YaeJ actively promote the nonrotated state during translation termination.
• Directed conformational changes, triggered by specific codon recognition, are essential for efficient termination and ribosome recycling.
• These findings integrate structural and dynamic data to explain ribosome rescue mechanisms.