Elongation Factor Tu D138N, a Mutant with Modified Substrate Specificity, as a Tool To Study Energy Consumption in Protein Biosynthesis

Abstract

Substitution Aspl38→Asn changes the substrate specificity of elongation factor (EF) Tu from GTP to XTP [Hwang & Miller (1987) J. Biol. Chem. 262, 13081-13085]. This mutated EF-Tu (EF-Tu D138N) was used to show that 2 XTP molecules are hydrolyzed for each elongation cycle [Weijland & Parmeggiani (1993) Science 259, 1311-1313]. Here we extend the study of the properties of this EF-Tu mutant and its function in the elongation process. In poly(U)-directed poly(phenylalanine) synthesis, the number of peptide chains synthesized using EF-Tu D138N-XTP was 30% higher than with EF-Tu wild type (wt)•GTP. However, since in the former case the average peptide chain length was correspondingly reduced, the number of the residues incorporated turned out to be nearly the same in both systems. The K′d values of the XTP and XDP complexes of EF-Tu D138N were similar to those of the GTP and GDP complexes of EF-Tu wt. The extent of leucine misincorporation and the kirromycin effect were also comparable to those in the EF-Tu wt/GTP system. The hydrolysis of two XTP molecules, very likely as part of two EF-Tu D138N-XTP complexes, for each elongation cycle was found to be independent of (i) MgCl2 concentration, (ii) ribosome concentration, and (iii) temperature (5–40 °C). With rate-limiting amounts of XTP the K′m of its XTPase activity corresponded to the K′m for XTP of poly(phenylalanine) synthesis (0.3–0.6 µM). This correlation strongly suggests that both XTP molecules are involved in the basic mechanism of the EF-Tu-mediated binding of aminoacyl-tRNA to the ribosome and do not participate in idling activities. With concentrations of MgCl2 higher than 9 mM, the EF-G-dependent GTPase became strongly uncoupled from poly(phenylalanine) synthesis, whereas the XTPase activity of EF-Tu started to be uncoupled at MgCl2 concentrations higher than 12 mM. The results of this work prove that the EF-Tu D138N/XTP system is a powerful tool for analyzing bioenergetic aspects of protein biosynthesis. © 1994, American Chemical Society. All rights reserved.