Acidic ribosomal proteins from eukaryotic cells. Effect on ribosomal functions

Area of Science:

• Molecular Biology
• Biochemistry

Background:

• Ribosomes are crucial for protein synthesis.
• Ethanol precipitation is a method to study ribosome structure and function.
• Specific ribosomal proteins play key roles in ribosomal activity.

Purpose of the Study:

• To investigate the effect of ethanol precipitation on Saccharomyces cerevisiae ribosome activity.
• To identify which ribosomal proteins are released under specific ethanol and NH4Cl concentrations.
• To determine the functional impact of released acidic proteins on ribosome activity.

Main Methods:

• Ethanol precipitation of yeast and rat liver ribosomes.
• Fractionation of released proteins based on acidity.
• Assays for polymerizing activity and elongation-factor-2-dependent GTP hydrolysis.
• Reconstitution experiments with released acidic proteins.
• Immunological cross-reaction studies.

Main Results:

• Ethanol precipitation at 0.4 M NH4Cl released acidic proteins from yeast and rat liver ribosomes.
• At 1 M NH4Cl, additional non-acidic proteins were lost.
• Release of acidic proteins caused minor inactivation of polymerizing activity but significantly affected GTP hydrolysis.
• Added acidic proteins restored GTPase activity but not polymerizing activity.
• Eukaryotic acidic proteins could not substitute for bacterial L7 and L12.
• No immunological cross-reactivity was found between bacterial and eukaryotic acidic proteins or among eukaryotic acidic proteins.

Conclusions:

• Acidic ribosomal proteins are essential for optimal GTPase activity in eukaryotic ribosomes.
• The functional roles of acidic proteins in eukaryotes differ from bacterial L7 and L12.
• Ethanol precipitation is a valuable tool for dissecting ribosome protein function.