Contribution of Arg288 of Escherichia Coli Elongation Factor Tu to Translational Functionality

Abstract

The recently solved structure of the ternary complex formed between GTP-bound elongation factor Tu and aminoacylated tRNA reveals that the elements of aminoacyl-tRNA that interact with elongation factor Tu can he divided into three groups: the T stem: the 3'-end CCA-Phe; and the 5' end. The conserved residues Arg2X8, LysX9 and Asn90 are involved in the binding of the 5' end. In the active, GTP-bound form of the elongation factor, Arg288 and Am90 are involved in the forination of a network of hydrogen bonds connecting the switch regions I and I1 of domain 1 with the rest of the molecule. This network is disrupted upon formation of the ternary complex. Arg288 was replaced by alanine, isoleucine, lysine or glutamic acid, and the resulting mutants have been subjected to an in vitro characterisation with the aim of clarifying the function of Arg288. Unexpectedly, the mutants behaved like the wild-type factor with regard to thc association and dissociation o f guanine nucleotides, and the intrinsic GTPasc activities are unchanged. Furthermore, the mutants were as efficient as the wild-type factor i n carrying out protein synthesis in vitro in the presence of an excess of aminoacyl-tRNA. However. the mutants' abilities to bind aminoacyl-tRNA and protect the labile arninoacyl bond were impaired, especially where the charge had been reversed.

Recognition and selection of tRNA in translation

“…3). The intrinsic arginine residue (Arg58), that is located in the switch I region of EF-Tu in a position homologous to that of the catalytic arginine in G a proteins, is not essential for GTP hydrolysis [27], and an extensive mutagenesis search did not identify any other side chains important for GTP hydrolysis in Escherichia coli EF-Tu [27][28][29][30].…”

Mutational analysis of Glu272 in elongation factor 1A of E. coli

“…Native PAGE can re£ect very subtle interactions ranging from a complete absence of binding to strong formation of the ternary complex, so it is very suitable for revealing the extent of the interactions between EF1A and aa-tRNA. These di¡erent degrees of interaction can be seen for example in our previous reports [16,17].…”

Mutagenesis of Arg335 in Bovine Mitochondrial Elongation Factor Tu and the Corresponding Residue in the Escherichia coli Factor Affects Interactions with Mitochondrial Aminoacyl-tRNAs

“…Arg288 is an important residue that stabilizes the binding of the 5' end of the aa-tRNA to EF-Tu. 22 Mutation of Arg288 reduces the formation of the ternary complex significantly. Removal of the 5' phosphate on the aa-tRNA allows ternary complex formation but reduces the efficiency of the aa-tRNA in polymerization.…”

“…The ability of wild-type and variant EF-Tu to protect Phe-tRNA Phe from spontaneous deacylation was monitored as described. 21,22 Wild-type and variant EF-Tu mt (1.0 µM) containing 10 µM GDP were incubated with either E. coli or mitochondrial [ 14 C]Phe-tRNA Phe (0.40 µM) at 0˚C for 20 min in a 100 µL reaction mixture containing 50 mM Tris-HCl (pH 7.8), 1 mM dithiothreitol, 71 mM KCl, 6.5 mM MgCl 2 , 2.5 mM phosphoenol pyruvate, 1.0 unit pyruvate kinase and 0.5 mM GTP. The reactions were shifted to 20˚C and aliquots (10 µL) were withdrawn at various times, precipitated in 5% trichloroacetic acid and treated as described above.…”

“…The dissociation of the ternary complex was monitored as described. 22,25 Wild-type and variant E. coli EF-Tu (1.08 µM and 1.02 µM respectively) containing 10 µM GDP were incubated in a 100 µL reaction mixture containing 50 mM Tris-HCl (pH 7.8), 1 mM dithiothreitol, 71 mM KCl, 6.5 mM MgCl 2 , 2.5 mM phosphoenol pyruvate, 1.0 unit pyruvate kinase and 0.5 mM GTP at 37˚C for 2.5 h to regenerate GTP in the EF-Tu samples. Immediately following this incubation, 10 µL of each sample was removed and the percentage of active EF-Tu molecules was determined as described above to ensure that activity was not lost during the incubation.…”