Studies of 30 S Escherichia coli ribosome reassembly using individual proteins labeled with an environmentally sensitive fluorescent probe
| Autor: | Kuei-Huang Huang, Charles R. Cantor |
|---|---|
| Rok vydání: | 1975 |
| Předmět: |
Ribosomal Proteins
Binding Sites Quenching (fluorescence) Protein subunit Ribosomal RNA Biology medicine.disease_cause Ribosome Fluorescence Models Structural Kinetics Spectrometry Fluorescence Biochemistry Structural Biology Covalent bond Ribosomal protein Escherichia coli medicine Ribosomes Molecular Biology Mathematics Fluorescent Dyes Protein Binding |
| Zdroj: | Journal of Molecular Biology. 97:423-441 |
| ISSN: | 0022-2836 |
| Popis: | Covalent fluorescent derivatives of a number of 30 S Escherichia coli ribosomal proteins have been prepared with 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole. These will successfully reassemble back into active 30 S ribosomes with normal sedimentation properties. Direct monitoring of the time course of fluorescence permits an analysis of the kinetics of reassembly with four individual labeled proteins, S5, S7, S8, S9. The assembly is apparent first-order. Similar rate constants were obtained in all four cases suggesting that the rate-limiting step is a co-operative folding or organization of many proteins. The effect of deleting various proteins on the fluorescence of reassembled 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole-containing particles was studied. Results suggest that S10 protein plays a critical role in determining acquisition of proper structure in the neighborhood of S5. Reassociation of fluorescent labeled 30 S particles with 50 S to form 70 S ribosomes occurred with little or no fluorescence change in all cases examined. However, marked effects were seen when iodide quenching was used to examine the reassociation. As one might expect, quite a few 30 S proteins are rendered less accessible to collisional quenching by solute ions in the presence of the 50 S. However two proteins, S16 and S20 appear to be more accessible in the 70 S particle than in the free 30 S subunit. This adds to the growing body of evidence that association of ribosomal subunits involves a substantial reorganization of the 30 S particle. |
| Databáze: | OpenAIRE |
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