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Partial reconstitution of active eukaryotic ribosomes following dissociation with dimethylmaleic anhydride
Abstract:
Modification of yeast ribosomes with dimethylmaleic anhydride, a reagent for protein amino groups, is accompanied by loss of polypeptide-synthesizing activity. This activity can be recovered by incubation at pH 6, which produces regeneration of the modified amino groups. Dimethylmaleic anhydride modification also causes the dissociation of proteins from the ribosomes. Protein-deficient ribosomal particles are prepared from 80 S ribosomes or 60 S subunits by treatment with a molar excess of reagent relative to ribosomal particles equal to 6300 or 4800, respectively. The core particles from 80 S ribosomes lack 18% and those from 60 S subunits 40% of the total protein in the corresponding untreated control. In both cases, there is a selective release of proteins, the protein-deficient particles being able to reconstitute active 60 S subunits upon addition of the corresponding split proteins. The reconstituted ribosomal particles, when assayed in the presence of native 40 S subunits, are active in poly(U)-directed polyphenylalanine synthesis (30-90% of the activity of a dimethylmaleic anhydride-untreated control, as compared to 0-15% when the split proteins are excluded). This procedure could prove useful in the study of the structure and function of the eukaryotic ribosome.
Insights
Yeast ribosomes lose protein synthesis ability when modified with dimethylmaleic anhydride, but this can be restored. This chemical modification selectively removes proteins, creating core particles that can be reconstituted into active ribosomal subunits.
Area of Science:
- Molecular Biology
- Biochemistry
- Cell Biology
Background:
- Ribosomes are essential for protein synthesis.
- Understanding ribosome structure and function is crucial for cell biology.
- Chemical modification offers a tool to probe macromolecular complexes.
Purpose of the Study:
- To investigate the effects of dimethylmaleic anhydride modification on yeast ribosomes.
- To develop a method for creating protein-deficient ribosomal particles.
- To assess the functional recovery of reconstituted ribosomal subunits.
Main Methods:
- Modification of yeast ribosomes and subunits with dimethylmaleic anhydride.
- Preparation of protein-deficient ribosomal core particles.
- Reconstitution of ribosomal subunits with removed proteins.
- Assay of polypeptide synthesis activity in reconstituted particles.
Main Results:
- Dimethylmaleic anhydride modification inactivated yeast ribosomes and caused protein dissociation.
- Selective removal of 18-40% of proteins yielded core ribosomal particles.
- Reconstitution with removed proteins restored significant polypeptide synthesis activity (30-90%).
Conclusions:
- Dimethylmaleic anhydride is a useful reagent for selectively removing ribosomal proteins.
- This method allows for the study of eukaryotic ribosome structure and function.
- Reconstitution experiments highlight the importance of specific proteins for ribosomal activity.