In vitro protein synthesis by the moderate halophile Vibrio costicola: site of action of Cl- ions.

AUTOR(ES)

Choquet, C G

RESUMO

In vitro protein synthesis in Vibrio costicola [poly(U)-directed incorporation of phenylalanine] was studied. The extent of protein synthesis was limited by the number of ribosomes present. Density gradient centrifugation experiments suggested that, after runoff of ribosomes from the artificial messenger, the 50S subunit was unable to attach to the 30S-messenger complex. As shown previously (M. Kamekura and D. J. Kushner, J. Bacteriol. 160:385-390, 1984), Cl- ions inhibited protein synthesis; indeed, the highest rate of synthesis took place in the lowest attainable Cl- concentration (37 mM). The inhibitory effects were partly reversed by glutamate and betaine, both of which are concentrated within cells of V. costicola. The strongest reversal was seen when both glutamate and betaine were present. Cl- ions can prevent binding of ribosomes to poly(U) and displace ribosomes already bound to this artificial messenger. The effects of Cl- ions on binding were also reversed by glutamate and betaine. Cl- ions did not affect accuracy of translation; they were shown previously (Kamekura and Kushner, J. Bacteriol. 160:385-390, 1984) not to affect phenylalanyl-tRNA synthetase. It was also found that washing ribosomes with inhibitory NaCl concentrations did not interfere with their ability to carry out protein synthesis later in optimal (low) salt concentrations. On the contrary, these ribosomes were more active than before they were washed. We conclude that the main site of action of Cl- in the system studied is on the binding of ribosomes to the mRNA.

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