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G-418, an elongation inhibitor of 80 S ribosomes
Abstract:
The mode of action of the aminoglycoside G-418 was studied in wheat-germ, cell-free translation systems programmed with rat-liver polyadenylated RNA. Incorporation of amino acids into protein was effectively inhibited by G-418 in the microM concentration range. The inhibition pattern obtained was not uniform. The synthesis of polypeptides with higher molecular weights was more inhibited than that of smaller polypeptides. An identical inhibition pattern within a similar range of concentrations was obtained with cycloheximide, a known elongation inhibitor. Translation activity was abolished when the wheat-germ 80 S ribosomes were removed and could be partially reconstructed upon addition of the ribosomes. Incubation with G-418 prior to isolation yielded ribosomes defective in their reconstruction ability. The inhibition pattern was not uniform and exhibited again the same relationship between the size of a polypeptide and the extent of inhibition of its synthesis. Therefore, we suggest that in wheat-germ, cell-free translation systems G-418 affects the 80 S ribosomes and inhibits the elongation cycle.
Insights
The aminoglycoside G-418 inhibits protein synthesis in wheat germ cell-free systems by affecting 80S ribosomes. This drug specifically hinders the elongation cycle, impacting larger polypeptide production more significantly.
Area of Science:
- Molecular Biology
- Biochemistry
- Protein Synthesis
Background:
- Aminoglycoside antibiotics, such as G-418, are known for their antimicrobial properties.
- Understanding the precise molecular mechanisms of these compounds is crucial for their effective application and for developing new therapeutic agents.
- Cell-free translation systems provide a powerful tool to dissect specific steps in protein synthesis.
Purpose of the Study:
- To elucidate the mode of action of the aminoglycoside G-418 in eukaryotic translation.
- To investigate the effect of G-418 on protein synthesis in a wheat germ cell-free system.
- To determine whether G-418 targets specific components or processes within the translation machinery.
Main Methods:
- Utilized wheat germ cell-free translation systems programmed with rat-liver polyadenylated RNA.
- Assessed the incorporation of amino acids into proteins at varying concentrations of G-418.
- Compared the inhibition pattern of G-418 with that of cycloheximide, a known elongation inhibitor.
- Investigated the role of wheat germ 80S ribosomes by their removal and subsequent addition.
Main Results:
- G-418 inhibited amino acid incorporation into protein in the micromolar concentration range.
- The inhibition was non-uniform, with a greater effect on the synthesis of higher molecular weight polypeptides.
- This pattern mirrored the effect of cycloheximide, suggesting an inhibition of the elongation cycle.
- Wheat germ 80S ribosomes were essential for translation, and pre-incubation with G-418 rendered them defective.
Conclusions:
- G-418 acts on the 80S ribosomes in wheat germ cell-free translation systems.
- The drug inhibits the elongation cycle of protein synthesis.
- The differential inhibition based on polypeptide size supports a role in elongation rather than initiation.