RESUMO
BACKGROUND: Ribosome recycling factor (RRF), in concert with elongation factor EF-G, is required for disassembly of the post-termination complex of a ribosome after the release of polypeptides. How RRF dissociates the complex has long been puzzling. Crystal structures of RRF molecules have been solved recently and shown to mimic a transfer RNA (tRNA) shape, which prompted us to examine whether RRF binds to the ribosome as tRNA does. RESULTS: The formation of ribosome complexes on the surface-coupled RRF and elongation factor EF-G of Escherichia coli was monitored in real time with a BIACORE 2000 instrument based on the surface plasmon resonance technique. RRF interacted with 70S ribosomes as well as 50S and 30S subunits, although it interacted preferentially with 50S subunits, which was clearly seen under high but physiological ionic conditions. This 50S interaction was diminished by a single amino acid substitutions for Arg132 of RRF, which did not appreciably affect the protein folding but nullified the activity in vivo and in vitro. Moreover, a set of antibiotics that inhibited the RRF-50S interaction were also inhibitory to the polysome breakdown activity of RRF in vitro. The BIACORE technique also worked very well in demonstrating the action of the antibiotics thiostrepton and fusidic acid, which are inhibitory to the RRF function by freezing the pre- and post-translocation intermediates catalysed by EF-G. CONCLUSIONS: These results suggest that the preferential interplay of RRF with the 50S subunit may be of biological significance, probably reflecting the mode of RRF action. The BIACORE technique proved useful for real-time monitoring of the interaction between the ribosome and translation factors, as well as for screening of potential inhibitors for ribosome recycling factor.
Assuntos
Escherichia coli/metabolismo , Fator G para Elongação de Peptídeos/metabolismo , Proteínas/metabolismo , Ribossomos/metabolismo , Ressonância de Plasmônio de Superfície/métodos , Substituição de Aminoácidos/genética , Aminoglicosídeos , Antibacterianos/farmacologia , Arginina/genética , Transporte Biológico Ativo/efeitos dos fármacos , Transporte Biológico Ativo/genética , Ácido Fusídico/farmacologia , Glicina/genética , Histidina/genética , Mutagênese Sítio-Dirigida , Polirribossomos/efeitos dos fármacos , Polirribossomos/genética , Polirribossomos/metabolismo , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/genética , Inibidores da Síntese de Proteínas/farmacologia , Estabilidade de RNA/efeitos dos fármacos , Estabilidade de RNA/genética , RNA Ribossômico/metabolismo , Proteínas Ribossômicas , Ribossomos/efeitos dos fármacos , Ribossomos/genética , Tioestreptona/farmacologiaAssuntos
Animais Domésticos/microbiologia , Animais Selvagens/microbiologia , Infecções por Campylobacter/veterinária , Campylobacter/isolamento & purificação , Portador Sadio/veterinária , Animais , Infecções por Campylobacter/epidemiologia , Campylobacter fetus/isolamento & purificação , Portador Sadio/epidemiologia , Reservatórios de Doenças , Fezes/microbiologia , JapãoRESUMO
Zinc ion, Cu2+ and Cd2+ at 10(-4) M or lower concentrations caused a marked decrease in the membrane potential of mucous epithelial cells in the isolated newt stomach without appreciable change in the effective membrane resistance and electrical coupling ratio. Cysteine and acetylpenicillamine antagonized the action of these ions. These ions as well as ouabain blocked the hyperpolarization of membrane due to an increase in intracellular Na+. The effect of the ions on the relationship between external K+-concentration and membrane potential was similar to that of ouabain. Discussion is made on the possibility that the ions inhibit active ion transport by combining with a SH-group of the cell membrane and, thus, bring about a decrease in membrane potential.
Assuntos
Mucosa Gástrica/efeitos dos fármacos , Potenciais da Membrana/efeitos dos fármacos , Metais/farmacologia , Animais , Cisteína/farmacologia , Condutividade Elétrica , Células Epiteliais , Epitélio/efeitos dos fármacos , Mucosa Gástrica/citologia , Técnicas In Vitro , Penicilamina/análogos & derivados , Penicilamina/farmacologia , SalamandridaeRESUMO
The membrane potential of mucous epithelial cells of isolated newt stomach was -23 mV on the average at 7 degrees C. The potential decreased when the temperature was lowered further, the normal external solution was replaced with K+-free solution, or ouabain or DNP was applied. The decreased membrane potential level of the cells in K+-free solution was not changed any more by adding ouabain to the solution. The membrane potential increased transiently beyond the control level when the stomach was returned to the normal solution after being exposed to isotonic NaCl solution with EGTA. The transient potential increase was blocked by ouabain. These results suggest that, even at such a low temperature as 7 degrees C, the active ion transport mechanism(s) of mucous epithelial cells in the newt stomach is still capable of functioning to some extent.
