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 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 degrees 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 microM).(ABSTRACT TRUNCATED AT 250 WORDS)
Weijland
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A
SDI 61840 du CNRS, Laboratoire de Biochemie, Ecole Polytechnique, Palaiseau, France.
Parlato
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G
Parmeggiani
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A
D004734:Energy Metabolism; D004926:Escherichia coli; D020963:GTP Phosphohydrolase-Linked Elongation Factors / Q000378:metabolism; D066298:In Vitro Techniques; D016297:Mutagenesis, Site-Directed; D010441:Peptide Chain Elongation, Translational; D020653:Peptide Elongation Factor G; D010444:Peptide Elongation Factor Tu / Q000378:metabolism*; D010445:Peptide Elongation Factors / Q000378:metabolism; D012265:Ribonucleotides / Q000378:metabolism; D012270:Ribosomes / Q000378:metabolism; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D013696:Temperature
D020653:Peptide Elongation Factor G; D010445:Peptide Elongation Factors; D012265:Ribonucleotides; C065326:xanthosine 5'-triphosphate; D020963:GTP Phosphohydrolase-Linked Elongation Factors; D010444:Peptide Elongation Factor Tu