Determination of the crystal structure of the ternary complex formed between elongation factor Tu:GTP and aminoacylated tRNA revealed three regions of interaction between elongation factor Tu and tRNA. The structure indicates that the conserved glutamic acid at position 271 in Thermus aquaticus EF-Tu could be involved in the binding of the 3' CCA-Phe end of the aminoacylated tRNA. Therefore, the corresponding residue, Glu259, of Escherichia coli EF-Tu was mutated into alanine, aspartic acid, glutamine and tyrosine, in order to substantiate the crystallographic structural evidence and to obtain further knowledge of the importance of this residue. All of the mutated proteins showed nucleotide binding properties similar to the wild type. In addition the GTPase activities were similar to the wild type. The mutation of Glu259 to either alanine or aspartic acid resulted in a reduced strength of interaction with tRNA, while mutation to tyrosine abolished completely the interaction with tRNA. Finally, mutation to glutamine resulted in an elongation factor Tu variant behaving like the wild type. In conclusion, the environment around the site binding the CCA-Phe end of the tRNA is very restricted spatially and chemically so that only a residue with almost the same size and chemical properties as glutamic acid fulfils the requirements with regard to size, salt bridge-formation potential and maintenance of the backbone conformation at the 259 position.

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.

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Although proteins and nucleic acids consist of different chemical components, proteins can mimic structures and possibly also functions of nucleic acids. Recently, structural mimicry was observed between two elongation factors in bacterial protein biosynthesis leading to the introduction of the concept of macromolecular mimicry. Macromolecular mimicry has further been proposed among initiation and release factors, thereby adding a new element to the description of protein synthesis in bacteria. Such mimicry has also been observed in other biological processes such as autoimmunity, DNA repair, and gene regulation, at both transcriptional and translational levels.

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