Histidine‐118 of elongation factor Tu: its role in aminoacyl‐tRNA binding and regulation of the GTPase activity

Abstract

The function of His"* in elongation factor (EF)-Tu from Escherichia coli was investigated by its substitution with glycine. The substitution had a differential effect on individual functions of the protein. The affinity for aminoacyl (aa)-tRNA and the intrinsic GTPase activity of the mutant EF-Tu were decreased whereas the response of its GTPase center to aa-tRNA was strongly increased. These results suggest that the region around Hisi's is involved in the binding of aa-tRNA and in the transmission of a turn-off signal generated by the interaction with aa-tRNA and directed to the GTPase center of EF-Tu.

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.…”

Mutants of EF‐Tu defective in binding aminoacyl‐tRNA

“…It is known from previous work that substitutions of the conserved residue His 118 to Ala or to Glu [9] or to Gly [10] in E. coli EF-Tu also cause significant decreases in the affinity of EF-Tu for aa-tRNA. His us is in the immediate structural neighbourhood of Leu 12° and Gin 124 in the interface of domains I and III.…”

“…Interestingly, the substitution HisllSGly also alters the interaction between guanine nucleotide and EF-Tu, decreasing EF-Tu's intrinsic GTPase activity in the absence, but increasing it in the presence of aatRNA [10]. Thus, substitutions in the domain interface, many of which apparently decrease the affinity of aa-tRNA for EFTu, are in some cases also associated with alterations in the interaction between EF-Tu and the bound guanine nucleotide.…”

“…Site-directed mutagenesis of conserved His residues in domain I (His 66 and His 118) show that alterations at these positions reduce the affinity of EF-Tu for aa-tRNA [9,10]. The residues Lys 2°s and Lys 23z in domain II of EF-Tu have been crosslinked, in the presence of kirromycin, to 3' oxidised tRNA [11].…”

The Importance of P-loop and Domain Movements in EF-Tu for Guanine Nucleotide Exchange

“…The flip of the P-loop was found in a number of other G-proteins in complexes with their respective guanine nucleotide exchange factors, suggesting that displacement of the P-loop is a universally important step in guanine nucleotide release (10). However, the contribution of EF-Tsinduced rearrangements in the P-loop of EF-Tu to the acceleration of nucleotide exchange remained unclear despite several mutagenesis studies (9,(11)(12)(13). In the present work, we examine the contribution of P-loop movement to nucleotide exchange by replacing one of the critical His residues, His-118, with Ala or Glu.…”