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[Rate of ribosome movement along messenger RNA in E. coli under normal and inhibited translation]
Abstract:
Considerable decrease of polysome number (22% as compared with 48% in normally grown culture) was observed under methionine starvation of E. coli Hfr (Met-)culture . At the same time the amount of 70S ribosomes increased up to 32%, while it was 2--6% in the control, the content of free ribosome subunits (50S+30S) being stable. The number of polysomes was the same (congruent to 50%) both in the control culture and under inhibition of protein synthesis in E. coli Hfr(Met-) cells with chloramphenicol, the content of 70S ribosomes was increased (30%) like in the case of methionine starvation, and the amount of free ribosome subunits was decreased (24% as compared with 46% in the control). The rate of ribosome movement in polysomes in the presence of chloramphenicol is comparable with that in the control. The rate of ribosome movement along mRNA under methionine starvation in 1.6 times lower than in normally grown E.coli culture. The level of (14)C-leucine incorporation into newly synthesizing polysome proteins under chloramphenicol inhibition of protein synthesis and methionine starvation comprised 20% and 12% of the incorporation level inthe control respectively. It suggested that ribosomes under inhibition of protein synthesis by chloramphenicol or amino acid starvation continue their movement along mRNA with the rate comparable with that in the control. However in this case no peptide bonds are formed ("abortive" translocation).
Insights
Methionine starvation and chloramphenicol inhibit protein synthesis in E. coli, causing polysome reduction and increased 70S ribosomes. Ribosomes continue moving along mRNA, but peptide bond formation is blocked, leading to "abortive" translocation.
Area of Science:
- Molecular Biology
- Bacteriology
- Biochemistry
Background:
- Ribosome function is crucial for protein synthesis.
- Amino acid starvation and protein synthesis inhibitors disrupt cellular processes.
Purpose of the Study:
- To investigate the effects of methionine starvation and chloramphenicol on E. coli ribosome dynamics.
- To understand the mechanism of "abortive" translocation during inhibited protein synthesis.
Main Methods:
- Culturing E. coli Hfr (Met-) under methionine starvation.
- Treating E. coli with chloramphenicol to inhibit protein synthesis.
- Analyzing ribosome distribution (polysomes, 70S ribosomes, subunits) via ultracentrifugation.
- Measuring the rate of ribosome movement along mRNA.
- Quantifying (14)C-leucine incorporation into newly synthesized proteins.
Main Results:
- Methionine starvation significantly decreased polysome number and increased 70S ribosomes in E. coli.
- Chloramphenicol treatment also increased 70S ribosomes and decreased free ribosome subunits, similar to starvation.
- Ribosome movement along mRNA was reduced under methionine starvation but comparable to control with chloramphenicol.
- (14)C-leucine incorporation was significantly lower under both conditions compared to control.
Conclusions:
- Ribosomes can continue moving along mRNA even when peptide bond formation is inhibited.
- Both amino acid starvation and chloramphenicol induce a state of "abortive" translocation in E. coli ribosomes.
- This suggests a regulatory mechanism decoupling mRNA translocation from peptide synthesis.