Evidence for a Negative Cooperativity between eIF5A and eEF2 on Binding to the Ribosome.

Area of Science:

• Molecular Biology
• Protein Synthesis
• Ribosome Function

Background:

• The eukaryotic translation initiation factor 5A (eIF5A) is unique for containing the amino acid hypusine.
• eIF5A plays critical roles in both translation initiation and elongation, particularly at ribosome stalling sequences like poly-proline.
• Understanding the interplay between eIF5A, tRNA, and elongation factor 2 (eEF2) on the ribosome is crucial for deciphering protein synthesis regulation.

Purpose of the Study:

• To elucidate the functional interaction between eIF5A, tRNA, and eEF2 on the ribosome surface.
• To quantify the binding affinity of eIF5A to ribosomal complexes and assess the impact of hypusine and eEF2 on this interaction.
• To investigate the consequences of impaired eIF5A-ribosome binding on translation and cell viability.

Main Methods:

• Fluorescence anisotropy assays were employed to measure the binding affinities of eIF5A to ribosomal subunits and complexes.
• The effects of charged tRNA (tRNAiMet) and eEF2 on eIF5A binding were assessed using these assays.
• Conditional yeast eIF5A mutants were utilized to study the functional consequences of altered ribosome binding.

Main Results:

• eIF5A binds directly and exclusively to the 60S ribosomal subunit in a hypusine-dependent manner, with significantly higher affinity than its lysine-containing form.
• eIF5A binding to the 80S ribosome exhibits positive cooperativity with P-site tRNA binding.
• eEF2 binding to the ribosome impairs eIF5A binding affinity, indicating negative cooperativity, and high-copy eEF2 is toxic to mutants with impaired eIF5A binding, causing translation elongation defects.

Conclusions:

• The study clarifies the direct, hypusine-dependent interaction of eIF5A with the 60S ribosomal subunit and its cooperative binding with tRNA.
• Negative cooperativity between eEF2 and eIF5A binding to the ribosome suggests a regulatory mechanism involving conformational changes.
• Impairment of the eIF5A-eEF2 interplay compromises translation elongation and cell growth, highlighting the importance of this interaction for cellular homeostasis.