Sordarin bound eEF2 unlocks spontaneous forward and reverse translocation on CrPV IRES

Area of Science:

• Molecular Biology
• Biochemistry
• Structural Biology

Background:

• Intergenic Region Internal Ribosome Entry Sites (IGR IRESs) of Discistroviridae initiate protein synthesis independently of canonical initiation factors.
• Elongation factor 2 (eEF2)-mediated IRES translocation represents a unique factor-catalyzed step in translation.
• Understanding ribosome conformation dynamics during IRES-mediated translation is crucial for deciphering non-canonical protein synthesis.

Purpose of the Study:

• To investigate the conformational dynamics of eukaryotic ribosomes during cricket paralysis virus IRES (CrPV IRES) translation at the single-molecule level.
• To elucidate the role of elongation factor 2 (eEF2) in promoting both forward and reverse translocation during IRES-mediated translation.
• To determine the effect of the antifungal sordarin on eEF2-ribosome interactions and IRES translocation efficiency.

Main Methods:

• Development of a single-molecule observation system by labeling ribosomal RNA (rRNA).
• Monitoring of ribosome conformation and translation dynamics using the developed system.
• Investigating the concentration-dependent effects of eEF2 on ribosome translocation.
• Analyzing the impact of sordarin on eEF2-ribosome complexes and IRES translocation.

Main Results:

• Pre-translocation 80S-IRES ribosomes predominantly adopted a semi-rotated conformation, with spontaneous exchange between rotated and non-rotated states.
• eEF2 promoted both forward and reverse ribosome translocation in an eEF2 concentration-dependent manner.
• Sordarin stabilized eEF2 on the ribosome in an extended conformation, enabling multiple translocation rounds per binding event.
• In the presence of sordarin, IRES translocation proceeded without GTP hydrolysis or phosphate release, suggesting thermally driven movements.

Conclusions:

• eEF2 actively promotes both forward and reverse translocation during CrPV IRES-mediated translation.
• Sordarin significantly enhances IRES translocation by stabilizing eEF2 and facilitating ribosomal conformational changes.
• The mid and late stages of CrPV IRES translocation, particularly in the presence of sordarin, are likely thermally driven processes unlocked by eEF2.