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Effect of the antibiotic purpuromycin on cell-free protein-synthesizing systems
F Rambelli1, M Brigotti, M Zamboni
1Dipartimento di Patologia sperimentale dell'Università di Bologna, Italy.
Abstract:
Purpuromycin, an antibiotic isolated from the culture broth of Actinoplanes ianthinogenes, which is very active against Gram-positive bacteria and fungi, inhibits protein synthesis in both prokaryotic and eukaryotic cell-free systems. The ID50 was 9 microM with the endogenous mRNA-directed rabbit reticulocyte lysate, 17 microM with a poly(U)-directed system from Escherichia coli and 69 microM with a poly(U)-directed system from Artemia salina cysts. Of the three steps of elongation, purpuromycin does not affect the peptidyl-transferase reaction, inhibits the elongation factor 1 (EF-1) dependent binding of phenylalanyl-tRNA and stimulates the GTP-dependent binding of EF-2. When protein synthesis is stopped by the addition of purpuromycin, the nascent peptide chains are found in the puromycin-reactive P site. The results suggest that the mechanism of action of purpuromycin is similar to that of fusidic acid. Both antibiotics would seem to produce a stable guanine nucleotide-ribosome-EF-2 complex which allows one round of translocation but prevents, because of a common or overlapping ribosomal binding site for the two elongation factors, the subsequent EF-1-dependent binding of aminoacyl-tRNA.
Insights
Purpuromycin, an antibiotic, inhibits protein synthesis by interfering with elongation factors. Its mechanism resembles fusidic acid, forming a stable complex that blocks further aminoacyl-tRNA binding.
Area of Science:
- Microbiology
- Molecular Biology
- Biochemistry
Background:
- Purpuromycin is an antibiotic from Actinoplanes ianthinogenes.
- It exhibits activity against Gram-positive bacteria and fungi.
- Purpuromycin inhibits protein synthesis in both prokaryotic and eukaryotic systems.
Purpose of the Study:
- To elucidate the mechanism of action of purpuromycin.
- To compare purpuromycin's inhibitory effects with other protein synthesis inhibitors.
Main Methods:
- Investigated purpuromycin's effect on cell-free protein synthesis systems (rabbit reticulocyte lysate, Escherichia coli, Artemia salina).
- Assessed inhibition of specific elongation steps: peptidyl-transferase, elongation factor 1 (EF-1) dependent tRNA binding, and elongation factor 2 (EF-2) dependent GTP binding.
- Analyzed the location of nascent peptide chains upon inhibition.
Main Results:
- Purpuromycin inhibits protein synthesis with varying potency across different systems (ID50: 9-69 microM).
- It does not affect peptidyl-transferase but inhibits EF-1 dependent phenylalanyl-tRNA binding.
- Purpuromycin stimulates EF-2 dependent GTP binding and traps nascent peptides in the P site.
Conclusions:
- Purpuromycin's mechanism involves creating a stable ribosome-EF-2 complex, similar to fusidic acid.
- This complex allows one translocation round but prevents subsequent EF-1 dependent aminoacyl-tRNA binding due to overlapping ribosomal sites.
- The antibiotic effectively halts protein synthesis by disrupting elongation factor interactions.