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Reversible modification of 50S ribosomal subunits with dimethylmaleic anhydride: protein-deficient particles
Abstract:
The reversible modification of protein amino groups with dimethylmaleic anhydride, which had already been used to dissociate proteins from the 70S ribosomes of Escherichia coli (Pintor-Toro, J. A., et al. (1979) Biochemistry 18, 3219) was applied to the preparation of protein-deficient particles from the 50S subunits. Three successive cycles of treatment with dimethylmaleic anhydride, separation of dissociated proteins and regeneration of the modified amino groups produce partially inactivated ribosomal 'cores' lacking proteins L7, L11 and L12, and having very small amounts of L1, L6 and L10. Incubation of these 'cores' with the corresponding split proteins is accompanied by complete reactivation of the polypeptide synthesizing activity as compared with control 50S subunits.
Insights
Researchers developed a method using dimethylmaleic anhydride to create protein-deficient ribosomal cores. These cores, when reconstituted with specific proteins, fully restored polypeptide synthesis activity, demonstrating a key role for these proteins in ribosome function.
Area of Science:
- Molecular Biology
- Biochemistry
- Ribosome research
Background:
- Ribosomes are essential for protein synthesis.
- Understanding the role of individual ribosomal proteins is crucial for deciphering translation mechanisms.
- Previous work demonstrated dimethylmaleic anhydride's utility in dissociating proteins from 70S ribosomes.
Purpose of the Study:
- To prepare protein-deficient particles from 50S ribosomal subunits.
- To investigate the role of specific ribosomal proteins in polypeptide synthesis.
- To assess the functional recovery of reconstituted ribosomal cores.
Main Methods:
- Reversible modification of protein amino groups using dimethylmaleic anhydride.
- Sequential treatment cycles to remove and then regenerate proteins.
- Preparation of protein-deficient ribosomal 'cores' from 50S subunits.
- Reconstitution of 'cores' with specific split proteins.
Main Results:
- Successfully generated partially inactivated ribosomal 'cores' lacking specific proteins (L7, L11, L12) and reduced amounts of others (L1, L6, L10).
- Reconstitution of these 'cores' with the corresponding split proteins led to complete reactivation of polypeptide synthesizing activity.
- The reconstituted activity matched that of control 50S subunits, indicating successful functional recovery.
Conclusions:
- Specific ribosomal proteins (L7, L11, L12) are essential for the full polypeptide synthesizing activity of 50S ribosomal subunits.
- Dimethylmaleic anhydride is an effective tool for selectively removing and regenerating ribosomal proteins.
- This method provides a valuable approach for studying ribosome structure-function relationships.