Anneke Talens

Specific alterations of the EF-Tu polypeptide chain considered in the light of its three-dimensional structure.

Specific alterations of the elongation factor Tu (EF‐Tu) polypeptide chain have been identified in a number of mutant species of this elongation factor. In two species, Ala‐375, located on domain II, was found by amino acid analysis to be replaced by Thr and Val, respectively. These replacements substantially lower the affinity of EF‐Tu.GDP for the antibiotic kirromycin. Since kirromycin can be cross‐linked to Lys‐357, also located on domain II but structurally very far from Ala‐375, these data suggest that the replacements alter the relative position of domains I and II. The Ala‐375 replacements also lower the dissociation rates of the binary complexes EF‐Tu.GTP and the binding constants for EF‐Tu.GTP and Phe‐tRNA. It is conceivable that these effects are also mediated by movements of domains I and II relative to each other. Replacement of Gly‐222 by Asp has been found in another mutant by DNA sequence analysis of the cloned tufB gene, coding for this mutant EF‐Tu. Gly‐222 is part of a structural domain, characteristic for a variety of nucleotide binding enzymes. Its replacement by Asp does not abolish the ability of EF‐Tu to sustain protein synthesis. It increases the dissociation rate of EF‐Tu.GTP by approximately 30%. In the presence of kirromycin this mutant species of EF‐Tu.GDP does not bind to the ribosome, in contrast to its wild‐type counterpart. A possible explanation is now open for experimental verification.

Novel RNA interactions with the elongation factor EF-Tu: consequences for protein synthesis and tuf gene expression

Abstract The elongation factor EF-Tu, discovered more than 20 years ago, has not suffered from a lack of attention. Despite numerous investigations, this important protein continues to yield new and interesting information about its functions, its three-dimensional structure and the organization and regulation of its two encoding genes.

Novel Functions of EF-Tu during Polypeptide Synthesis and tuf Gene Expression

The function of the elongation factor EF-Tu during protein synthesis is intimately related to that of tRNA. Interestingly, one of the EF-Tu encoding genes and four tRNA genes are cotranscribed and form one operon (Lee et al., 1981). During the elongation cycle, EF-Tu forms a ternary complex with aminoacyl-tRNA and GTP that interacts with the ribosome/ mRNA complex (Miller and Weissbach, 1977). Recently, we discovered that as a result of this interaction, a second tRNA binding site is induced on the factor protein that can accommodate not only aminoacyl-tRNA but also peptidyl-tRNA and nonacylated tRNA with relatively high affinities (Van Noort et al., 1982). Mutant species of EF-Tu have been isolated in our laboratory (Van de Klundert et al., 1978) that are altered in their tRNA binding and in their GTPase center. Mutant Escherichia coli cells harboring these EF-Tu species were found to suppress nonsense mutations. Studies of the regulation of tufA and tufB, the two EF-Tu encoding genes, indicated that EF-Tu is an autogenous repressor of tufB expression.

The interaction between aminoacyl-tRNA and the mutant elongation factors Tu AR and B0.

The binding of Tyr-[AEDANS-s2C]tRNA(Tyr) (Tyr-tRNA(Tyr) modified at the penultimate cytidine residue with a thio group at position 2 of the pyrimidine ring, to which an N-(acetylaminoethyl)-5-naphthylamine-1-sulfonic acid fluorescence group is attached) to mutant elongation factor (EF)-Tu species from E. coli, EF-TuAR (Ala-375----Thr) and EF-TuBO (Gly-222----Asp), both complexed to GTP, was investigated in absence of kirromycin by measuring the change in fluorescence of the modified tRNA induced by complex formation. The calculated dissociation constant in the case of EF-TuAR is about 4 nM and in the case of EF-TuB0, about 1 nM. These values are higher than that of wild-type EF-Tu, which was 0.24 nM measured with the same system. The affinity between either EF-TuB0.kirromycin.GDP or EF-TuB0.kirromycin.GTP on the one hand, and a mixture of aminoacyl-tRNAs on the other, was measured with zone-interference gel electrophoresis. The dissociation constants are 20 microM and 7 microM, respectively, a factor of about two higher than in the case of wild-type EF-Tu.kirromycin. These findings provide a clue for the observed increase in translational errors in strains carrying the mutations. Furthermore, the experiments with EF-TuB0.kirromycin deepen our understanding of the effects of the B0 mutation on the kirromycin phenotype of the mutant cells concerned.

The Elongation Factor EF-Tu FROM E. Coli Activates The tRNA-Tufb Operon in Trans by Binding To A Cis-Acting Region Upstream of The Promoter

A highly representative member of the group of guanine nucleotide binding proteins is the polypeptide chain elongation factor Tu of E. coli (EF-Tu). This translational factor is a multifunctional protein able to bind, beside GDP and GTP, a relatively large number of ligands, such as tRNA, ribosomes, the elongation factor EF-Ts and antibiotics like kirromycin and pulvomycin. EF-Tu is also involved in the replication of RNA phages as one of the host donated subunits of the viral RNA replicase (Miller et al., 1977; Bosch et al., 1983; Bosch et al., 1986). EF-Tu therefore is an attractive object for studies of the relationship between structure and function. Considerable progress has been made with the elucidation of the three-dimensional structure of EF-Tu as is reported elsewhere in this volume (Jurnak et al., 1989; Nyborg et al., 1989). Genetic studies revealed that EF-Tu is encoded by two genes: tufA and tufB, located some 660 kbp apart on the E. coli chromosome (Jaskunas et al., 1975). The two genes have been cloned and sequenced (An and Friesen, 1980; Yokota et al., 1980; Hudson et al., 1981) so that a firm experimental basis has been laid for structure/function studies. They are part of two operons quite different in character (compare Fig. 1).