Kinetics and thermodynamics of the interaction of elongation factor Tu with elongation factor Ts, guanine nucleotides, and aminoacyl-tRNA

G. Romero, Vincent Chau, R. L. Biltonen

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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 kinetics procedures. The kinetics of the exchange reaction were found to be consistent with the formation of a ternary complex EF-Tu·GTP·EF-Ts. The equilibrium association constants of EF-Ts to the EF-Tu·GTP complex and of GTP to EF-Tu·EF-Ts were calculated to be 7 x 107 and 2 x 106 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 constants from the EF-Tu·GTP complex (2.5 x 10-2 s-1). A procedure based on the observation that EF-Tu·GTP protects the aminoacyl-tRNA molecule from phosphodiesterase I-catalyzed hydrolysis was used to study the interactions of EF-Tu·GTP with Val-tRNA(Val) and Phe-tRNA(Phe). Binding constants of Phe-tRNA(Phe) and Val-tRNA(Val) to EF-Tu·GTP of 4.8 x 107 and 1.2 x 107 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.

Original languageEnglish (US)
Pages (from-to)6167-6174
Number of pages8
JournalJournal of Biological Chemistry
Volume260
Issue number10
StatePublished - Jan 1 1985

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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