A STUDY OF ENZYMIC TRANSFER OF AMINOACYL-RNA TO ESCHERICHIA COLI RIBOSOMES

Three fractions, F-IA, F-IB, and F-II, have been obtained from extracts of Es-cherichia coli W by chromatography on DEAE-Sephadex. The guanosine 5′-triphosphate (GTP)-dependent transfer of aminoacyl-RNA to ribosomes is catalyzed by either F-IA or F-IB, and polypeptide synthesis is catalyzed by either fraction in the presence of F-II. Fraction F-IB is more labile than F-IA. Both F-IA and F-IB interact with GTP to form guanine nucleotide-enzyme complexes that are retained by Millipore filters. In the presence of aminoacyl-RNA, F-IA and F-IB interact with GTP to form a complex that is not retained by a Millipore filter. Guanosine 5′-diphosphate (GDP) also interacts with F-IA or F-IB to form a complex; however, no subsequent interaction with aminoacyl-RNA is observed. Both the GTP-dependent enzymic transfer of aminoacyl-RNA to ribosomes and the interaction of enzyme with GTP are inhibited by GDP. The enzymic transfer of aminoacyl-RNA to ribosomes is stimulated by NH4+ and a sulfhydryl compound, and the interaction of the guanine nucleotide-enzyme complex with aminoacyl-RNA is also stimulated by NH4+. Deacylated sRNA, which inhibits nonenzymic binding, has no significant effect on the enzymic binding of aminoacyl-RNA to ribosomes. The guanine nucleotide-aminoacyl-RNA complex formed by the interaction of GTP, phenylalanyl-RNA, and either F-IA, F-IB, or a mixture of the two can be recovered by filtration through a Millipore filter and chromatography on Sephadex G-25. The active fractions from the Sephadex G-25 column contain close to stoichiometric amounts of phenylalanine and the guanine moiety and the -γ-phosphate moiety of the GTP. The amount of phenylalanine transferred to the ribosomes from the complex is 2-fold greater than the amount transferred from phenylalanyl-RNA alone and is equivalent to the amount transferred from phenylalanyl-RNA in the presence of enzyme and GTP. The guanine moiety of the complex is also transferred to the ribosomes, but no significant transfer of the γ-phosphate of the GTP present in the active fractions is observed. © 1968.