Abstract
The exchange of elongation factor Tu (EF-Tu)-bound GTP in the presence and absence of elongation factor Ts (EF-Ts) was monitored by equilibrium exchange kinetic procedures. The kinetics of the exchange reaction were found to be consistent with the formation of a ternary complex EF-Tu X GTP X EF-Ts. The equilibrium association constants of EF-Ts to the EF-Tu X GTP complex and of GTP to EF-Tu X EF-Ts were calculated to be 7 X 10(7) and 2 X 10(6) M-1, respectively. The dissociation rate constant of GTP from the ternary complex was found to be 13 s-1. This is 500 times larger than the GTP dissociation rate constant from the EF-Tu X GTP complex (2.5 X 10(-2) s-1). A procedure based on the observation that EF-Tu X GTP protects the aminoacyl-tRNA molecule from phosphodiesterase I-catalyzed hydrolysis was used to study the interactions of EF-Tu X GTP with Val-tRNAVal and Phe-tRNAPhe. Binding constants of Phe-tRNAPhe and Val-tRNAVal to EF-Tu X GTP of 4.8 X 10(7) and 1.2 X 10(7)M-1, respectively, were obtained. The exchange of bound GDP with GTP in solution in the presence of EF-Ts was also examined. The kinetics of the reaction were found to be consistent with a rapid equilibrium mechanism. It was observed that the exchange of bound GDP with free GTP in the presence of a large excess of the latter was accelerated by the addition of aminoacyl-tRNA. On the basis of these observations, a complete mechanism to explain the interactions among EF-Tu, EF-Ts, guanine nucleotides, and aminoacyl-tRNA has been developed.
Highlights
The exchangeof elongation factor Tu (EF-Tu)-bound initiated ribosome promoting the binding of aa-tRNA to the GTP in the presence anadbsence of elongation factor A site of the ribosome (1-3)
A procedure based on the observation that EF-Tu. ciates with a rate constant of about s-’ (10).Since the in GTPprotects the aminoacyl-tRNA molecule from vivo rate of peptide synthesis is of the order of 10 amino phosphodiesterase I-catalyzed hydrolysis was used to acids/s/ribosome,the spontaneous rate of dissociation of study the interactioonfsEF-Tu
Our experiments show that the substrate supply reaction proceeds via the formation of several intermediates according to thereaction scheme shown below: Tu and Ts, the guanine nucleotides GDP and GTP, and the several aminoacyl-tRNA species required for the peptide synthesis process
Summary
From the Departments of Biochemistry and Pharmacology, University of Virginia School of Medicine, Charlottesville, Virginia22908. A procedure based on the observation that EF-Tu. ciates with a rate constant of about s-’ (10).Since the in GTPprotects the aminoacyl-tRNA molecule from vivo rate of peptide synthesis is of the order of 10 amino phosphodiesterase I-catalyzed hydrolysis was used to acids/s/ribosome (ll),the spontaneous rate of dissociation of study the interactioonfsEF-Tu. GTP wiVthal-tRNAVd the EF-Tu GDPcomplex is much too slow to account for any and Phe-tRNAPh”.Binding constants of Phe-tRNAPhe and Val-tRNAVdto EF-Tu-GTPof 4.8 X lo7and 1.2 X lo7M-’, respectively, were obtained. Our experiments show that the substrate supply reaction proceeds via the formation of several intermediates according to thereaction scheme shown below: Tu and Ts, the guanine nucleotides GDP and GTP, and the several aminoacyl-tRNA species required for the peptide synthesis process. After 5 min of incubation, 2 0 4 aliquots of the reaction mixture were absorbed onto
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