Effects of antibiotics, N-acetylaminoacyl-tRNA and other agents on the elongation-factor-Tu dependent and ribosome-dependent GTP hydrolysis promoted by 2'(3')-O-L-phenylalanyladenosine

Abstract

GTP hydrolysis on elongation factor (EF) Tu . ribosome complexes has been assayed in the presence of 2'(3')-O-L-phenylalanyladenosine (AdoPhe), i.e. the 3'-terminal portion of Phe-tRNAPhe. Several requirements of the reaction have been characterized. Maximal activity is observed at 60-120 mM NH4Cl and 5-15 mM magnesium acetate. The reaction requires the free sulfhydryl group of EF-Tu normally implicated in aminoacyl-tRNA binding. Intact EF-Tu cannot be replaced by a large tryptic fragment of EF-Tu (Mr 39,000) that retains the ability to bind guanosine nucleotides. The aminoglycoside antibiotics, neomycin C and several kanamycins and gentamicins, stimulate the AdoPhe-promoted GTPase. Surprisingly, however, other closely related antibiotics, like neomycin B, paromomycin and ribostamycin, are ineffectual, thus indicating subtle differences in the actions of these antibiotics. AcPhe-tRNAPhe, bound to the ribosomal A-site, stimulates the AdoPhe-promoted GTPase, but this compound or AcTyr-tRNATyr, present in unbound form, strongly inhibits the reaction. These results suggest that N-blocked aminoacyl-tRNAs form ternary complexes with EF-Tu . GTP, which have not been previously detected because of their low stability.