The exchange of ribosomal subunits during the release of growing polypeptide chains by puromycin has been investigated in a bacterial cell-free system engaged in protein synthesis. The addition of sperinidine, used as a stabilizing agent of 70S monomers, caused a strong inhibition of the subunit exchange. This result led us to conclude that upon premature release of unfinished protein chains by the antibiotic, the ribosomes fall off mRNA as 70S particles. This behavior is different from that occurring during physiological termination of translation, where the ribosomes detach in a dissociated form. Some implications of the postulated mechanism are also discussed.Puromycin is a well-known inhibitor of protein synthesis in vivo as well as in cell-free systems. Its structure, analogous to aminoacyl-tRNA, leads to the premature release of unfinished polypeptide chains as polypeptidyl-puromycin derivatives (1-4).However, the fate of ribosomes upon release of growing protein chains by puromycin is still not well understood. Schlessinger et al. (5) reported that ribosomes engaged in polyphenylalanine synthesis were dissociated into subunits after treatment with puromycin, because the free 30S-50S couples were unstable. More recently Kohler et al. (6) could not confirm these results; they found that 708 particles bearing polyphenylalanyl-tRNA remained intact when peptide chains were liberated by the antibiotic.Both experiments lead to opposite conclusions, but neither of them can be used as a good model of the puromycin reaction at the polysomal level, which constitutes a more physiological system.
Paromomycin, an aminoglycoside antibiotic having low mammalian cell toxicity, is one of the drugs currently used in the chemotherapy of cutaneous and visceral leishmaniasis. In order to understand the mode of action of this antibiotic at the molecular level, we have investigated the effects of paromomycin on protein synthesis in Leishmania and its mammalian hosts. We were able to demonstrate that in vivo protein synthesis in the promastigote stage of the parasite and its proliferation rate are markedly inhibited by paromomycin while being only slightly affected by other aminoglycoside antibiotics, such as streptomycin and neomycin B. Furthermore, both in vitro polypeptide synthesis induced by poly(U) as mRNA and accuracy of translation are significantly decreased by paromomycin in cell-free systems containing ribosomal particles of Leishmania promastigotes. Conversely, when ribosomes from mammalian cells are used instead of the protozoan particles, polyphenylalanine synthesis is only barely reduced by the antibiotic and the translation misreading remains almost unaltered. Surface plasmon resonance analysis of the interaction between paromomycin and protozoan or mammalian cell ribosomal RNAs shows a strong binding of antibiotic to the parasite ribosomal decoding site and practically no interaction with the mammalian cell counterpart. Our results indicating differential effects of paromomycin on the translation processes of the Leishmania parasite and its mammalian hosts can explain the therapeutic efficiency of this antibiotic as an antileishmaniasis agent.
Mammalian ornithine decarboxylase (ODC) is among the most labile of cellular proteins, with a half-life of usually less than an hour. Like other short-lived proteins ODC is degraded by the 26S proteasome. Its degradation is not triggered by ubiquitination, but is stimulated by the binding of an inducible protein, antizyme. Truncations and mutations in the C terminus of mammalian ODC have been shown to prevent the rapid turnover of the enzyme, demonstrating the presence of a degradation signal in this region. Moreover, ODCs from the trypanosomatid parasites Trypanosoma brucei and Leishmania donovani, which lack this Cterminal domain, are metabolically stable, and recombination of T. brucei ODC with the C terminus of mammalian ODC confers a short half-life to the fusion protein when expressed in mammalian cells. In the present study we have cloned and sequenced the ODC gene from the trypanosomatid Crithidia fasciculata. To our knowledge, this is the first protozoan shown to have an ODC with a rapid turnover. The sequence analysis revealed a high homology between C. fasciculata ODC and L. donovani ODC, despite the difference in stability. We demonstrate that C. fasciculata ODC has a very rapid turnover even when expressed in mammalian cells. Moreover, ODC from C. fasciculata is shown to lack the C-terminal degradation domain of mammalian ODC. Our findings indicate that C. fasciculata ODC contains unique signals, targeting the enzyme for rapid degradation not only in the parasite but also in mammalian cells.
The effect of streptomycin on polypeptide synthesis in vivo and in vitro has been investigated using polyamine auxotrophic mutants of Escherichia coli grown in the presence or in the absence of putrescine. We found that streptomycin caused a marked inhibition of protein synthesis in polyamine-supplemented cells whereas bacteria starved for polyamines were less sensitive to the action of the antibiotic. Neomycin, kanamycin and kasugamycin had a behaviour similar to streptomycin while spectinomycin, gentamicin and tetracycline brought about a strong inhibition of protein synthesis both in polyamine-starved and unstarved bacteria.The increase of misreading induced by the addition of streptomycin in vitro was higher in extracts derived from bacteria cultivated in the presence of polyamines. This effect was observed in cell-free systems of streptomycinsensitive and resistant strains. In contrast, spermidine added in virro caused an improvement in the accuracy of translation.Analysis of sodium dodecyl sulphate/polyacrylamide gel electrophoresis of the labelled polypeptides synthesized in vivo seems to indicate that the starvation for polyamine or the presence of streptomycin may lead to premature termination with the appearance of unfinished peptide chains.Polyamines are present in all living cells and have been implicated in a wide variety of metabolic processes [I -41.The isolation of some polyamine auxotrophic bacterial mutants [5, 61 has furnished a very convenient system for the study of the physiological role of these strongly basic substances. It has been previously reported that some Escherichia coli double mutants unable to synthesize putrescine (the precursor of spermidine) showed a decreased rate of growth and protein synthesis when starved for polyamines [7,8]. Studies carried out with cell-free systems obtained from these bacterial mutants grown in the absence or in the presence of putrescine have demonstrated that polyamines play a direct role in translation [9]. The decreased polypeptide synthesis observed in vitro with extracts from putrescine-depleted cells programmed either with poly(U) or natural messenger RNA is most probably due to defective 30-S particles [lo]. These abnormal 30-S subunits have a poor efficiency in the initiation steps of translation [Ill. Although there is not yet any direct proof, the elongation and termination processes of protein synthesis could also be affected.Recent investigations on protein synthesis in eukaryotic cell-free systems have shown that when magnesium was partially replaced by spermidine or spermine, the polypeptide elongation rate was increased with a concomitant reduction of the amount of short unfinished polypeptide chains [12,13]. This effect may be due to an acceleration of the elongation process and also to an improvement in the fidelity of the recognition step which decreases the probability of misreading certain codons corresponding to different amino acids as termination signals [13]. These results and those obtained in bacteria indicating a n increase...
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