Effect of the antibiotic purpuromycin on cell-free protein-synthesizing systems

Rambelli, F; Brigotti, Maurizio; Zamboni, M; Denaro, Maurizio; Montanaro, Lucio; Sperti, S

doi:10.1042/bj2590307

Cited by 11 publications

(10 citation statements)

References 18 publications

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“…positioning of EF-Tu to allow this interaction to occur. This is consistent with the previously unexplained observation that the SRL plays a critical role in the binding and function of both EF-Tu and EF-G on the ribosome (53)(54)(55). Additionally, it is now clear how the ribosome, via the SRL, functions as a GTPase-activating protein for EF-Tu, stabilizing the transition state for GTP hydrolysis, similar to cellular GTPases and their catalytic partner proteins.…”

Section: Figuresupporting

confidence: 89%

Structural Basis of the Translational Elongation Cycle

Voorhees

Ramakrishnan

2013

Annu. Rev. Biochem.

261

278

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The sequential addition of amino acids to a growing polypeptide chain is carried out by the ribosome in a complicated multistep process called the elongation cycle. It involves accurate selection of each aminoacyl tRNA as dictated by the mRNA codon, catalysis of peptide bond formation, and movement of the tRNAs and mRNA through the ribosome. The process requires the GTPase factors elongation factor Tu (EF-Tu) and EF-G. Not surprisingly, large conformational changes in both the ribosome and its tRNA substrates occur throughout protein elongation. Major advances in our understanding of the elongation cycle have been made in the past few years as a result of high-resolution crystal structures that capture various states of the process, as well as biochemical and computational studies.

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Section: Figuresupporting

confidence: 89%

Structural Basis of the Translational Elongation Cycle

Voorhees

Ramakrishnan

2013

Annu. Rev. Biochem.

261

278

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“…Since Rambelli et al (1989) had reported that purpuromycin inhibited cell-free protein synthesis in Artemia salina and in rabbit reticulocyte systems, as well as in E. coli, we investigated the effect of purpuromycin on poly(U)-directed polyphenylalanine synthesis in a C. albicans cell-free system (Fig. 6).…”

Section: Cell-free Protein-synthesis Assaysmentioning

confidence: 99%

“…No data are available in the literature on the mechanism of action of purpuromycin in intact micro-organisms, although Rambelli et al (1989) have reported that it inhibited cell-free protein synthesis both in E. coli and in eukaryotic systems (rabbit reticulocytes and Artemia salina). We have now studied the mode of action of purpuromycin in cultures of B. subtilis and C. albicans.…”

Section: Introductionmentioning

confidence: 99%

Mechanism of action of purpuromycin

Landini¹,

Corti²,

Goldstein³

et al. 1992

Biochemical Journal

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Purpuromycin, an antibiotic active against both fungi and bacteria, shows different modes of action against these two kinds of micro-organisms; in Candida albicans it inhibits RNA synthesis, whereas in Bacillus subtilis protein synthesis is primarily affected, with DNA and RNA synthesis blocked at higher concentrations of the drug. In bacterial cell-free protein-synthesis systems, purpuromycin did not inhibit synthesis from endogenous mRNA (elongation of peptides initiated within the intact cell) but inhibited MS2-phase RNA-dependent protein synthesis (which requires initiation) by 50% at 0.1 mg/l. Poly(U)-directed polyphenylalanine synthesis was 50% inhibited by 20 mg of purpuromycin/l when added to a complete system; however, when purpuromycin was preincubated with ribosomes dissociated into 30 S and 50 S subunits, the concentration for 50% inhibition fell to 0.1 mg/l. By contrast, in a C. albicans cell-free system poly(U)-directed polyphenylalanine synthesis was partially inhibited only at 200 mg/l. Purpuromycin also inhibited polynucleotide synthesis in vitro in reactions using Escherichia coli or wheat-germ RNA polymerases or E. coli DNA polymerase I. We suggest that in bacteria the primary target of purpuromycin is on ribosomes and that its action precedes the elongation step of protein synthesis. The effect on nucleic acid synthesis in both fungi and bacteria may be due to interaction of purpuromycin with DNA.

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“…Purpuromycin inhibits protein synthesis in both prokaryotic and eukaryotic cell-free systems (42,43), and it is evident that the inhibition of translation is through the specific inhibition of aminoacyl–tRNA formation (41). Neomycin B inhibits the phenylalanylation of E. coli tRNA Phe ; one neomycin molecule is bound to the upper part of the anticodon stem in the yeast tRNA Phe –aminoglycoside crystal complex (40).…”

Section: Introductionmentioning

confidence: 99%

Pentamidine binds to tRNA through non-specific hydrophobic interactions and inhibits aminoacylation and translation

Sun

Zhang

2008

Nucleic Acids Research

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The selective and potent inhibition of mitochondrial translation in Saccharomyces cerevisiae by pentamidine suggests a novel antimicrobial action for this drug. Electrophoresis mobility shift assay, T1 ribonuclease footprinting, hydroxyl radical footprinting and isothermal titration calorimetry collectively demonstrated that pentamidine non-specifically binds to two distinct classes of sites on tRNA. The binding was driven by favorable entropy changes indicative of a large hydrophobic interaction, suggesting that the aromatic rings of pentamidine are inserted into the stacked base pairs of tRNA helices. Pentamidine binding disrupts the tRNA secondary structure and masks the anticodon loop in the tertiary structure. Consistently, we showed that pentamidine specifically inhibits tRNA aminoacylation but not the cognate amino acid adenylation. Pentamidine inhibited protein translation in vitro with an EC50 equivalent to that binds to tRNA and inhibits tRNA aminoacylation in vitro, but drastically higher than that inhibits translation in vivo, supporting the established notion that the antimicrobial activity of pentamidine is largely due to its selective accumulation by the pathogen rather than by the host cell. Therefore, interrupting tRNA aminoacylation by the entropy-driven non-specific binding is an important mechanism of pentamidine in inhibiting protein translation, providing new insights into the development of antimicrobial drugs.

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Effect of the antibiotic purpuromycin on cell-free protein-synthesizing systems

Cited by 11 publications

References 18 publications

Structural Basis of the Translational Elongation Cycle

Structural Basis of the Translational Elongation Cycle

Mechanism of action of purpuromycin

Pentamidine binds to tRNA through non-specific hydrophobic interactions and inhibits aminoacylation and translation

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