Yeast eukaryotic initiation factor 4B (eIF4B) enhances complex assembly between eIF4A and eIF4G in vivo.

Area of Science:

• Molecular Biology
• Protein Interactions
• Gene Expression Regulation

Background:

• The eukaryotic initiation factor 4F (eIF4F) complex, comprising eIF4E, eIF4G, and eIF4A, is essential for mRNA cap binding and translation initiation.
• The HEAT domain of eIF4G stabilizes the RNA helicase activity of eIF4A, a critical step in translation.
• The mechanism by which yeast eIF4B (yeIF4B/Tif3) stimulates translation, unlike its mammalian counterpart, remains unclear.

Purpose of the Study:

• To investigate the role of yeast eIF4B in the function of the eIF4F complex.
• To elucidate the mechanism by which yeIF4B stimulates translation initiation.
• To understand the interaction between yeIF4B, eIF4G, and eIF4A.

Main Methods:

• Site-directed mutagenesis of yeast eIF4G HEAT domains to create temperature-sensitive (Ts(-)) mutations.
• Genetic suppression studies involving overexpression of yeIF4B and eIF4A.
• In vivo and in vitro biochemical assays to analyze eIF4A·eIF4G complex formation and stability.

Main Results:

• Specific mutations in the eIF4G HEAT domain were suppressed by yeIF4B or eIF4A overexpression, while others required only eIF4A.
• yeIF4B overexpression restored native eIF4A·eIF4G complexes in vivo and rescued mutant complexes in vitro.
• yeIF4B enhances eIF4A binding to eIF4G, promoting eIF4F assembly.

Conclusions:

• Yeast eIF4B acts in vivo to promote eIF4F assembly.
• yeIF4B enhances a conformation of the yeast eIF4G HEAT domain that favors stable eIF4A binding.
• This mechanism is critical for efficient mRNA translation initiation in yeast.