Formation and Properties of T Factor Complexes

1. A modified procedure is presented for the isolation from Escherichia coli Q13 of the protein Factor T necessary for GTP-dependent polyuridylate-directed synthesis of polyphenylalanine. 2. Factor T forms a stable ternary complex with GTP and phenylalanyl-tRNA, as determined by gel filtration, but not with either one alone. Other nucleoside triphosphates cannot substitute for GTP in complex formation nor can N -formylmethionyl-tRNA or deacylated tRNA replace phenylalanyl-tRNA. Factor T also forms a complex with GDP, but this reaction is not stimulated by phenylalanyl-tRNA. 3. Competition and labeling studies reveal that the predominant species bound to membrane filters by T factor is GDP and not GTP. Label from 3 H-GTP is bound primarily as the result of hydrolysis to 3 H-GDP by a GTPase activity which is associated with the T factor preparation. In the absence of aminoacyl-tRNA, T-GTP-aminoacyl-tRNA is not retained on membrane filters. 4. Two complexes of T factor, T-GDP and T-GTP-Phe-tRNA, are interchangeable through a dynamic equilibrium. T-GTP*-Phe-tRNA + GDP ⇌ T-GDP + GTP* + Phe-tRNA 5. The ternary complex (T-GTP-Phe-tRNA) efficiently transfers phenylalanyl-tRNA to ribosomes in the presence of polyuridylate with the concomitant hydrolysis of GTP to GDP and P i ; this reaction is unaffected by fusidic acid. The GDP formed is bound to protein, presumably T factor. For every molecule of phenylalanyl-tRNA transferred to ribosomes, 1 molecule of GTP is hydrolyzed and 1 molecule of the resulting GDP is bound to protein.