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Polypeptide formation and polyribosomes in Escherichia coli treated with chloramphenicol
Abstract:
In Escherichia coli cultures maximally inhibited with chloramphenicol, formation of polypeptides still continued at a slow, constant rate for at least 90 min. The rate of leucine incorporation was reduced to 0.5%, but methionine was only reduced to 2%, suggesting that chains are normally initiated with methionine but are prematurely released at a short chain length. Consistent with this possibility was the distribution of the products on Sephadex columns: a range of peptides longer than 4 and shorter than 60 to 70 residues was seen. Less than 10% of the peptides broke down during a chase with cold amino acids, and during continuous labeling they accumulated progressively. On the average, one peptide was formed per ribosome every 5 min. Peptide synthesis in the presence of chloramphenicol was still dependent on ribosome translocation; it stopped in a mutant with an inactivated temperature-sensitive elongation factor G. But even in the absence of translocation, new messenger ribonucleic acid (mRNA) chains were found joined to one or a few ribosomes. The chains had a size distribution comparable to that of mRNA from polyribosomes of growing cells. They were stabilized for an average time of about 5 min, but were more rapidly degraded after puromycin was added to the cells. This suggests that stabilization may be related to the average time spent by a ribosome on an mRNA chain, with or without polypeptide formation.
Insights
Even with chloramphenicol inhibition, Escherichia coli polypeptide formation continues slowly. This suggests premature peptide chain release, impacting protein synthesis research.
Area of Science:
- Molecular Biology
- Bacteriology
Background:
- Chloramphenicol is a known inhibitor of protein synthesis.
- Understanding residual protein synthesis under inhibition is crucial for molecular biology research.
Purpose of the Study:
- To investigate the mechanism and characteristics of residual polypeptide formation in chloramphenicol-treated Escherichia coli.
- To determine the fate of messenger RNA (mRNA) and nascent peptides during inhibited protein synthesis.
Main Methods:
- Utilized chloramphenicol to maximally inhibit protein synthesis in Escherichia coli cultures.
- Measured amino acid incorporation (leucine, methionine) to assess polypeptide synthesis rates.
- Analyzed peptide product size distribution using Sephadex chromatography.
- Investigated the role of ribosome translocation and elongation factor G (EF-G).
- Examined mRNA stability and degradation kinetics in the presence and absence of translocation.
Main Results:
- Polypeptide formation persisted at a slow, constant rate for at least 90 minutes.
- Methionine incorporation was significantly less reduced than leucine incorporation, indicating premature chain release.
- Sephadex analysis revealed a distribution of peptides, predominantly short chains.
- Peptide synthesis remained dependent on ribosome translocation, ceasing in EF-G deficient mutants.
- New mRNA chains associated with ribosomes were observed even without translocation, with a size distribution similar to growing cells.
- mRNA stabilization was linked to ribosome association time, with faster degradation after puromycin treatment.
Conclusions:
- Chloramphenicol treatment leads to the synthesis of short, prematurely released peptides in Escherichia coli.
- Ribosome translocation is essential for continued, albeit slow, peptide synthesis under chloramphenicol.
- mRNA stabilization is associated with ribosome binding, suggesting a role in translation regulation even during inhibition.