Polypeptide chain initiation in eukaryotes: functional identity of supernatant factor from various sources

Area of Science:

• Molecular Biology
• Biochemistry
• Cell Biology

Background:

• Eukaryotic cells possess polypeptide chain initiation factors crucial for protein synthesis.
• These factors are analogous to prokaryotic initiation factor IF-2, promoting specific tRNA binding to ribosomes.

Purpose of the Study:

• To investigate the characteristics of eukaryotic polypeptide chain initiation factors.
• To compare these factors with prokaryotic initiation factor IF-2, particularly regarding GTP dependence and source.

Main Methods:

• Assaying the AUG-dependent binding of fMet-tRNA(f) to the small ribosomal subunit.
• Measuring the conversion of bound amino-acyl-tRNA to fMet-puromycin upon 60S subunit addition.
• Testing the sensitivity of these reactions to initiation inhibitors and SH-binding reagents.

Main Results:

• Eukaryotic initiation factors promote fMet-tRNA binding and fMet-puromycin formation without GTP requirement.
• Factors from Artemia, mouse fibroblasts, and rat liver supernatants, and reticulocyte ribosome washes, exhibit these activities.
• While supernatant factors resemble E. coli IF-2 in SH-reagent sensitivity, the reticulocyte factor differs.
• Factors are generally interchangeable between Artemia and rat liver ribosomes, but Artemia factor and E. coli IF-2 are not.

Conclusions:

• Eukaryotic polypeptide chain initiation factors share functional similarities with prokaryotic IF-2 but possess distinct regulatory mechanisms, notably GTP independence.
• The study identifies and characterizes these factors from various eukaryotic sources, highlighting both conserved and divergent properties.
• Cross-species and cross-ribosome experiments reveal specificities and broad applicability of these essential translational machinery components.