The role of Glu259 in Escherichia coli elongation factor Tu in ternary complex formation

Abstract

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.

C-terminal interaction of translational release factors eRF1 and eRF3 of fission yeast: G-domain uncoupled binding and the role of conserved amino acids

“…The eRF3C domain that is sufficient for binding to eRF1 does not include the G-domain motifs+ This is in sharp contrast with other translational G proteins, elongation factors EF-Tu and EF-1a, or initiation factors IF2 and eIF-2, whose aminoacyl-tRNA or N-formylmethionyl-tRNA binding is controlled by G-domain function: GTP stimulates the association and GDP dissociates the complex+ There have been numerous reports that the N-terminal domain, including the G domain, of EF-Tu and EF-1a plays a crucial role in the binding of aminoacyl-tRNA directly or indirectly: the binding is diminished by mutations of Lys-4 (Laurberg et al+, 1998), Arg-7 (Mansilla et al+, 1997), Lys-9 (Laurberg et al+, 1998), Arg-58 , Lys-89 (Wiborg et al+, 1996), Asn-90 (Wiborg et al+, 1996), Gly-94 , His-118 (Jonak et al+, 1994), and Glu-259 (Pedersen et al+, 1998) of E. coli EF-Tu; Thr-62 of T. thermophilus EF-Tu (Ahmadian et al+, 1995); and Gly-280 of Salmonella typhimurium EF-Tu (Tubulekas & Hughes, 1993)+ Some of these substitutions, however, are known to affect the stability of the GTP form of EF-Tu/EF-1a relative to the GDP form, and thereby diminish the binding of aminoacyl-tRNA+ Because of the functional requirement for continuous delivery of aminoacyl-tRNA during protein elongation, the G-domain activity influences, directly or indirectly, the binding of aminoacyl-tRNA+ On the other hand, guanine nucleotides do not seem to influence the eRF1-eRF3 interaction+ They form a complex in vitro both in the presence (Zhouravleva et al+, 1995) or absence (Stansfield et al+, 1995;Frolova et al+, 1998) of GTP+ Therefore, the G-domain function of eRF3 may not be to change the binding of eRF1, but instead to change the binding of the ribosome or to catalyze final translocation of the ribosome+ Once eRF3 is associated with eRF1 before or after binding to the ribosome, the two probably remain associated via their C-termini interaction until their release from the ribosome, showing a clear functional difference between eRF3 and EF-Tu/EF-1a+ FIGURE 6. Comparison of the amino acid sequences of eRF3s and elongation factors EF-Tu and EF-1a+ The similarity alignments of eRF1s were accomplished using the PILEUP program from the GCG program package (Devereux et al+, 1984)+ Identical and similar amino acids are boxed in black and gray, respectively+ Asterisks indicate amino acids of T. aquaticus EF-Tu that are involved in tRNA binding in the three-dimensional structure (Nissen et al+, 1996)+ Daggers represent amino acids of S. pombe eRF3 that were mutated to alanine+ The number refers to the amino acid position counted from the N-terminal Met+ FIGURE 7.…”

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

“…Native PAGE can reflect 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 different degrees of interaction can be seen for example in our previous reports [16, 17].…”

The Interface between Escherichia coli Elongation Factor Tu and Aminoacyl-tRNA

“…Indeed, k pep values reported for H66A and E378A EF-Tu were found to be identical to cthose of the wild-type protein 4,24 and N90A, E259A, and H66A have activities comparable to those of wild-type protein using a polyU directed translation assay. 27,29 …”