A single amino acid substitution in elongation factor Tu disrupts interaction between the ternary complex and the ribosome

Abstract

Elongation factor Tu (EF-Tu) GTP has the primary function of promoting the efficient and correct interaction of aminoacyl-tRNA with the ribosome. Very little is known about the elements in EF-Tu involved in this interaction. We describe a mutant form of EF-Tu, isolated in Salmonella typhimurium, that causes a severe defect in the interaction of the ternary complex with the ribosome. The mutation causes the substitution of Val for Gly-280 in domain II of EF-Tu. The in vivo growth and translation phenotypes of strains harboring this mutation are indistinguishable from those of strains in which the same tufgene is insertionally inactivated. Viable cells are not obtained when the other tuf gene is inactivated, showing that the mutant EF-Tu alone cannot support cell growth. We have confirmed, by partial protein sequencing, that the mutant EF-Tu is present in the cells. In vitro analysis of the natural mixture of wild-type and mutant EF-Tu allows us to identify the major defect of this mutant. Our data shows that the EF-Tu is homogeneous and competent with respect to guanine nucleotide binding and exchange, stimulation of nucleotide exchange by EF-Ts, and ternary complex formation with aminoacyl-tRNA. However various measures of translational efficiency show a significant reduction, which is associated with a defective interaction between the ribosome and the mutant EFTu. GTP aminoacyl-tRNA complex. In addition, the antibiotic kirromycin, which blocks translation by binding EF-Tu on the ribosome, fails to do so with this mutant EF-Tu, although it does form a complex with EF-Tu. Our results suggest that this region of domain II in EF-Tu has an important function and influences the binding of the ternary complex to the codon-programmed ribosome during protein synthesis. Models involving either a direct or an indirect effect of the mutation are discussed.The prokaryotic translation factor elongation factor Tu (EF-Tu) mediates the productive interaction of aminoacyltRNA (aa-tRNA) with the ribosome. The ternary complex EF-Tu. GTP. aa-tRNA interacts with the ribosome such that the anticodon region of aa-tRNA is properly positioned in the ribosomal A site on the 30S subunit, while the aa region remains bound to EF-Tu outside the A site (43). EF-Tu on the ribosome protects bases in the universally conserved a-sarcin loop of 23S rRNA on the 50S ribosomal subunit, suggesting that this may form at least part of its ribosomal binding site (20,44). This conclusion is supported by data showing that a base substitution mutation in the ot-sarcin loop can affect the binding of ternary complex to the ribosome (62). If the tRNA codon and the mRNA anticodon in the 30S A site match, there is a high probability that the GTP on EF-Tu will be hydrolyzed. Whether the probability of GTP hydrolysis depends mainly on the length of time that the ternary complex spends on the ribosome or on signals transmitted to EF-Tu after correct codon-anticodon interaction is an open question. However, after GTP hydrolysis, EF-Tu-GDP leaves the ribosome and is...

Limited Proteolysis and Amino Acid Replacements in the Effector Region ofThermus thermophilusElongation Factor Tu

“…A naturally occurring kirromycin-resistant mutant of E. coli has been described, in which Gly222 of the EF-Tu encoded by tujB is replaced by Asp (Duisterwinkel et al, 1981). This variant is affected in its interaction with the ribosome (Swart and Parmeggiani, 1987), as is an EF-Tu variant of Salnzonella typhirnurium with substitution of Val280 by Gly (Tubulekas and Hughes, 1993). Since both amino acids are located i n domain IT, it is assumed that the ribosome-induced conformational change of EF-Tu and GTPase stimulation are mediated by domain 11.…”

Properties of Isolated Domains of the Elongation Factor Tu from Thermus Thermophilus HB8

“…It is expected that the domains I1 and I11 in the case of EF-Tu are essential for interac-tions involved in polypeptide chain elongation, i.e. the interactions with aminoacyl-tRNA, the ribosome and the nucleotide exchange factor EF-Ts (Ott and Sprinzl, 1992;Peter et al, 1990a;Tubulekas and Hughes, 1993).…”

Structure-based sequence alignment of elongation factors Tu and G with related GTPases involved in translation

“…In the present alignment this region has two gaps that separate the sequence G379-K381 in EF-G. The corresponding glycine residue has been found to be important for ribosome interaction in Salmonella typhimurium EF-Tu (Tubulekas and Hughes 1993) and according to this alignment is conserved in several translation factors as was previously proposed UEvarsson et al 1994). This short region has a very similar conformation in EF-Tu and EF-G but the residues do not superimpose very closely in space when the rest of the domains are superimposed.…”