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[Preparation of Escherichia coli 70S ribosomes labeled with 35S]
Abstract:
[35S]--70S ribosomes (150 Ci/mmol) were isolated from E. coli MRE-600 cells grown on glucose-mineral media in the presence of [35S] ammonium sulfate. The labeled 30S and 50S subunits were obtained from [35S] ribosomes by centrifugation in a sucrose density gradient of 10--30% under dissociating conditions (0.5 mM Mg2+). The activity of [35S]--70S ribosomes obtained by reassociation of the labeled subunits during poly(U)-dependent diphenylalanine synthesis was not less than 70%. The activity of [35S]--70S ribosomes during poly(U)-directed polyphenylalanine synthesis was nearly the same as that of the standard preparation of unlabeled ribosomes. The 23S, 16S and 5S RNAs isolated from labeled ribosomes as total rRNA contained no detectable amounts of their fragments as revealed by polyacrylamide gel electrophoresis. The [35S] ribosomal proteins isolated from labeled ribosomes were analyzed by two-dimensional gel electrophoresis. The [35S] label was found in all proteins, with the exception of L20, L24 and L33 which did not contain methionine or cysteine residues.
Insights
Researchers successfully labeled E. coli 70S ribosomes using [35S] ammonium sulfate. These labeled ribosomes maintained high activity in protein synthesis, demonstrating their utility for studying ribosomal structure and function.
Area of Science:
- Molecular Biology
- Biochemistry
- Microbiology
Background:
- Ribosomes are essential molecular machines responsible for protein synthesis.
- Studying ribosome structure and function requires methods for labeling and tracking these complexes.
- Escherichia coli (E. coli) is a widely used model organism in molecular biology research.
Purpose of the Study:
- To develop and validate a method for radiolabeling E. coli 70S ribosomes with sulfur-35 ([35S]).
- To assess the integrity and functional activity of the labeled ribosomes in protein synthesis.
- To analyze the distribution of the [35S] label within ribosomal components (RNA and proteins).
Main Methods:
- E. coli MRE-600 cells were cultured in media containing [35S] ammonium sulfate for metabolic labeling.
- 70S ribosomes were isolated and dissociated into 30S and 50S subunits using sucrose density gradient centrifugation.
- Labeled subunits were reassociated to form functional 70S ribosomes.
- Ribosome activity was assessed using poly(U)-directed phenylalanine polymerization.
- Ribosomal RNA (rRNA) and proteins were analyzed by polyacrylamide gel electrophoresis and two-dimensional gel electrophoresis, respectively.
Main Results:
- High-activity [35S]-labeled 70S ribosomes were successfully prepared, with reassociation efficiency of labeled subunits exceeding 70%.
- The functional activity of labeled 70S ribosomes in polyphenylalanine synthesis was comparable to unlabeled ribosomes.
- Analysis confirmed the integrity of 23S, 16S, and 5S ribosomal RNAs.
- [35S] labeling was detected in most ribosomal proteins, with exceptions for L20, L24, and L33, which lack methionine and cysteine.
Conclusions:
- Metabolic labeling of E. coli 70S ribosomes with [35S] is feasible and yields functionally active particles.
- The developed method allows for the study of ribosomal protein distribution and integrity.
- This technique provides a valuable tool for investigating ribosome biogenesis, structure, and function.