ELONGATION-FACTOR TU D138N, A MUTANT WITH MODIFIED SUBSTRATE-SPECIFICITY, AS A TOOL TO STUDY ENERGY-CONSUMPTION IN PROTEIN-BIOSYNTHESIS

Substitution Asp138-->Asn changes the substrate specificity of elongat ion factor (EF) Tu from GTP to XTP [Hwang and Miller (1987) J. Biol. C hem. 262, 13081-13085]. This mutated EF-Tu (EF-Tu D138N) was used to s how that 2 XTP molecules are hydrolyzed for each elongation cycle [Wei jland & 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 for mer case the average peptide chain length was correspondingly reduced, the number of the residues incorporated turned out to be nearly the s ame 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-T u wt. The extent of leucine misincorporation and the kirromycin effect were also comparable to those in the EF-Tu wt/GTP system. The hydroly sis of two XTP molecules, very likely as part of two EF-Tu D138N.XTP c omplexes, for each elongation cycle was found to be independent of (i) MgCl2 concentration, (ii) ribosome concentration, and (iii) temperatu re (5-40 degrees C). With rate-limiting amounts of XTP the K'(m) of it s XTPase activity corresponded to the K'(m) for XTP of poly(phenylalan ine) synthesis (0.3-0.6 mu M). This correlation strongly suggests that both XTP molecules are involved in the basic mechanism of the EF-Tu-m ediated binding of aminoacyl-tRNA to the ribosome and do not participa te in idling activities. With concentrations of MgCl2 higher than 9 mM , the EF-G-dependent GTPase became strongly uncoupled from poly(phenyl alanine) synthesis, whereas the XTPase activity of EF-Tu started to be uncoupled at MgCl2 concentrations higher than 12 mM. The results of t his work prove that the EF-Tu D138N/XTP system is a powerful tool for analyzing bioenergetic aspects of protein biosynthesis.