Eukaryotic translation initiation factor 5 (eIF5) acts as a classical GTPase-activator protein

Area of Science:

• Molecular Biology
• Biochemistry
• Genetics

Background:

• GTP hydrolysis is crucial for mRNA translation initiation, elongation, and termination.
• The precise mechanism of GTP hydrolysis during translation, and the involvement of ribosomal components versus extrinsic factors, remained unclear.

Purpose of the Study:

• To investigate the role of eukaryotic initiation factor 5 (eIF5) in GTP hydrolysis during translation initiation.
• To determine if eIF5 functions as a GTPase-activating protein (GAP) and to identify key residues involved.

Main Methods:

• Utilized biochemical assays to assess GTP hydrolysis stimulation by eIF5.
• Performed site-directed mutagenesis on conserved arginine residues (Arg15 and Arg48) within eIF5.
• Evaluated the impact of mutations on eIF5's interaction with eIF2 and its ability to support in vitro translation.

Main Results:

• eIF5 demonstrated characteristics of a classical GAP, including enhanced interaction with eIF2 in the presence of AlF(4)(-).
• A conserved arginine residue (Arg15) in eIF5 was identified as critical for stimulating GTP hydrolysis and supporting translation.
• Mutation studies suggested a second arginine (Arg48) also contributes to the GTPase active site of the eIF2.eIF5 complex.

Conclusions:

• eIF5 acts as a classical GAP, indicating that GTP hydrolysis during translation involves extrinsic proteins, not solely ribosomal components.
• The findings elucidate a key mechanism in translation initiation and suggest other translation factors may also function as GAPs.