The elongation factor Tu.kirromycin complex has two binding sites for tRNA molecules

The interaction of the polypeptide chain elongation factor Tu (EF-Tu) with the antibiotic kirromycin and tRNA has been studied by measuring the extent of protein modification with N-tosyl-L-phenylalanine chloromethylketone (TPCK) and N-ethylmaleimide (NEM). Kirromycin protects both EF-Tu.GDP and EF-Tu.GTP against modification with TPCK. Binding of aminoacyl-tRNA added at increasing concentrations to a solution of 40 microM EF-Tu.GDP.kirromycin complex re-exposes the TPCK target site on the protein. However, when the aminoacyl-tRNA concentration is raised beyond 20 microM, TPCK labeling drops again and is blocked completely at approximately 300 microM aminoacyl-tRNA. By contrast, addition of uncharged tRNA or N- acetylaminoacyl -tRNA enhances TPCK labeling of the protein over the entire tRNA concentration range studied. These data strongly suggest that kirromycin induces in EF-Tu.GDP an additional tRNA binding site that can bind uncharged tRNA, aminoacyl-tRNA, and N- acetylaminoacyl -tRNA. Support for this assumption is provided by measuring the modification of EF-Tu.GDP with the sulfhydryl reagent NEM. Moreover, NEM modification also indicates an additional tRNA binding site on EF-Tu.GTP.kirromycin, which could not be detected with TPCK. Mapping of the tryptic peptides of EF-Tu.GDP labeled with [14C]TPCK revealed only one target site for this agent, i.e., cysteine-81. Modification occurred at the same site in the presence and in the absence of kirromycin and uncharged tRNA.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mutants of the elongation factor EF-Tu, a new class of nonsense suppressors

Read-through of nonsense codons has been studied in wild-type Escherichia coli cells and in cells harbouring mutant species of the elongation factor EF-Tu. The two phenomena differ essentially. Readthrough of UGA in wild-type cells is reduced by inactivation of tufB but is restored to the original level by introducing into the cell plasmid-borne EF-Tu. This shows that the natural UGA leakiness is dependent on the intracellular concentration of EF-Tu. Strains of E. coli harbouring mutant species of the elongation factor EF-Tu suppress the nonsense codons UAG, UAA and UGA. Suppression shows a codon context dependence. It requires the combined action of two different EF-Tu species: EF-TuAR(Ala 375----Thr) and EF-TuBo(Gly 222----Asp). Cells harbouring EF-TuAR(Ala 375----Thr) and wild-type EF-TuB, or wild-type EF-TuA and EF-TuBo(Gly 222----Asp) do not display suppressor activity. These data demonstrate that mutated tuf genes form an additional class of nonsense suppressors. The requirement for two different mutant EF-Tu species raises the question whether translation of sense codons also occurs by the combined action of two EF-Tu molecules on the ribosome.     

The structural and functional basis for the kirromycin resistance of mutant EF-Tu species in Escherichia coli.

A structural and functional understanding of resistance to the antibiotic kirromycin in Escherichia coli has been sought in order to shed new light on the functioning of the bacterial elongation factor Tu (EF-Tu), in particular its ability to act as a molecular switch. The mutant EF-Tu species G316D, A375T, A375V and Q124K, isolated by M13mp phage-mediated targeted mutagenesis, were studied. In this order the mutant EF-Tu species showed increasing resistance to the antibiotic as measured by poly(U)-directed poly(Phe) synthesis and intrinsic GTPase activities. The K'd values for kirromycin binding to mutant EF-Tu.GTP and EF-Tu.GDP increased in the same order. All mutation sites cluster in the interface of domains 1 and 3 of EF-Tu.GTP, not in that of EF-Tu.GDP. Evidence is presented that kirromycin binds to this interface of wild-type EF-Tu.GTP, thereby jamming the conformational switch of EF-Tu upon GTP hydrolysis. We conclude that the mutations result in two separate mechanisms of resistance to kirromycin. The first inhibits access of the antibiotic to its binding site on EF-Tu.GTP. A second mechanism exists on the ribosome, when mutant EF-Tu species release kirromycin and polypeptide chain elongation continues.     

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.     

Altered regulation of the guanosine 5'-triphosphate activity in a kirromycin-resistant elongation factor Tu

In the preceding article a mutant elongation factor Tu (EF-TuD2216) resistant to the action of kirromycin was found to display a spontaneous guanosine 5'-triphosphatase (GTPase) activity, i.e., in the absence of aminoacyl transfer ribonucleic acid (tRNA) and ribosome-messenger RNA. This is the first example of an Ef-Tu supporting GTPase activity in the absence of macromolecular effectors and/or kirromycin. In this study we show that this activity is elicited by increasing NH4+ concentrations. As additional effect, the mutation caused an increased affinity of EF-Tu for GTP. Ammonium dependence of the GTPase activity an increased affinity for GTP are two properties also found with wild-type EF-Tu in the presence of kirromycin [Fasano, O., Burns, W., Crechet, J.-B., Sander, G., & Parmeggiani, A. (1978) Eur. J. Biochem. 89, 557-565; Sander, G., Okonek, M., Crechet, J.-B., Ivell, R., Bocchini, V., & Parmeggiani, A. (1979) FEBS Lett. 98, 111-114]. Therefore, both binding of kirromycin to wild-type EF-Tu and acquisition of kirromycin resistance introduce functionally related modifications. Kirromycin at high concentrations (0.1 mM) does not interact with mutant EF-TuD2216.GDP but still does with EF-TuD2216.GTP in agreement with our previous finding that EF-Tu.GTP is the preferential target of the antibiotic in the wild type [Fasano, O., Bruns, W., Crechet, J.-B., Sander, G., & Parmeggiani, A. (1978) Eur. J. Biochem. 89, 557-565). The GTPase activity of mutant EF-Tu in the presence of aminoacyl-tRNA and ribosome.mRNA is much higher than with wild-type EF-Tu and also much less dependent on the presence of mRNA. Miscoding for leucine, measured as poly(U)-directed poly(phenyl-alanine/leucine) synthesis at increasing Mg2+ concentrations, is identical for both wild-type and mutant EF-Tu.     

Codon-dependent conformational change of elongation factor Tu preceding GTP hydrolysis on the ribosome.

The mechanisms by which elongation factor Tu (EF-Tu) promotes the binding of aminoacyl-tRNA to the A site of the ribosome and, in particular, how GTP hydrolysis by EF-Tu is triggered on the ribosome, are not understood. We report steady-state and time-resolved fluorescence measurements, performed in the Escherichia coli system, in which the interaction of the complex EF-Tu.GTP.Phe-tRNAPhe with the ribosomal A site is monitored by the fluorescence changes of either mant-dGTP [3'-O-(N-methylanthraniloyl)-2-deoxyguanosine triphosphate], replacing GTP in the complex, or of wybutine in the anticodon loop of the tRNA. Additionally, GTP hydrolysis is measured by the quench-flow technique. We find that codon-anticodon interaction induces a rapid rearrangement within the G domain of EF-Tu around the bound nucleotide, which is followed by GTP hydrolysis at an approximately 1.5-fold lower rate. In the presence of kirromycin, the activated conformation of EF-Tu appears to be frozen. The steps following GTP hydrolysis--the switch of EF-Tu to the GDP-bound conformation, the release of aminoacyl-tRNA from EF-Tu to the A site, and the dissociation of EF-Tu-GDP from the ribosome--which are altogether suppressed by kirromycin, are not distinguished kinetically. The results suggest that codon recognition by the ternary complex on the ribosome initiates a series of structural rearrangements resulting in a conformational change of EF-Tu, possibly involving the effector region, which, in turn, triggers GTP hydrolysis.     

Effects of the mutation glycine-222----aspartic acid on the functions of elongation factor Tu

We have studied the properties of a mutant elongation factor Tu, encoded by tufB (EF-TuBo), in which Gly-222 is replaced by Asp. For its purification from the kirromycin-resistant EF-Tu encoded by tufA (EF-TuAr), a method was developed by exploiting the different affinities to kirromycin of the two factors and the competition between kirromycin and elongation factor Ts (EF-Ts) for binding to EF-Tu. The resulting EF-TuBo kirromycin and EF-TuAr EF-Ts complexes are separated by chromatography on diethylaminoethyl-Sephadex A-50. For the first time we have succeeded in obtaining a tufB product in homogeneous form. Compared with wild-type EF-Tu, EF-TuBo displays essentially the same affinity for GDP and GTP, with only the dissociation rate of EF-Tu GTP being slightly faster. Protection of amino-acyl-tRNA (aa-tRNA) against nonenzymatic deacylation by different EF-Tu species indicates that conformational alterations occur in the ternary complex EF-TuBo GTP aa-tRNA. However, the most dramatic modification is found in the EF-TuBo interaction with the ribosome. Its activity in poly(Phe) synthesis as well as in the GTPase activity associated with the interaction of its ternary complex with the ribosome mRNA complex requires higher Mg2+ concentrations than wild-type EF-Tu (Mg2+ optimum at 10-14 vs. 6 mM), even if EF-TuBo can sustain enzymatic binding of aa-tRNA to ribosomes at low Mg2+. The anomalous behavior of EF-TuBo is reflected in a remarkable increase of the fidelity in poly(Phe) synthesis, especially at high Mg2+ concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interaction of elongation factor Tu from Escherichia coli with aminoacyl-tRNA carrying a fluorescent reporter group on the 3' terminus

Transfer ribonucleic acids containing 2-thiocytidine in position 75 ([s2C]tRNAs) were prepared by incorporation of the corresponding cytidine analogue into 3'-shortened tRNA using ATP(CTP):tRNA nucleotidyltransferase. [s2C]tRNA was selectively alkylated with fluorescent N-iodoacetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine (1,5-I-AEDANS) on the 2-thiocytidine residue. The product [AEDANS-s2C]aminoacyl-tRNA, forms a ternary complex with Escherichia coli elongation factor Tu and GTP, leading to up to 130% fluorescence enhancement of the AEDANS chromophore. From fluorescence titration experiments, equilibrium dissociation constants of 0.24 nM, 0.22 nM and 0.60 nM were determined for yeast [AEDANS-s2C]Tyr-tRNATyr, yeast Tyr-tRNATyr, and the homologous E. coli Phe-tRNAPhe, respectively, interacting with E. coli elongation factor Tu.GTP. The measurement of the association and dissociation rates of the interaction of [AEDANS-s2C]Tyr-tRNATyr with EF-Tu.GTP and the temperature dependence of the resulting dissociation constants gave values of 55 J mol-1 K-1 for delta S degrees' and -34.7 kJ mol-1 for delta H degrees' of this reaction.     

The effect of mutations in EF-Tu on its affinity for tRNA as measured by two novel and independent methods of general applicability

Elongation factor Tu is essential for binding and a correct delivery of aminoacyl-tRNA during protein biosynthesis. For a good characterization of its interaction with tRNA in terms of structure-function relationship, determinations of kinetic equilibrium parameters are of great value. We describe two novel methods for that purpose. One method is based on EF-Tu protection of the tRNA 3' acceptor end against RNase A cleavage and yields the Kd value together with the corresponding dissociation and association rate constants from one single set of experiments. The other is a rapid method for screening relative affinities of mutant EF-Tus for tRNA. It is based on competition between EF-Tu species with and without a (His)6 extension for the same aminoacyl-tRNA and yields a relative Kd value. The method can be of general importance for the measuring of ligand affinities of all sorts of His-tagged proteins. Both methods are illustrated by their application in the analysis of mutant EF-Tus with changed interactions with tRNA and antibiotics. Raising the assay temperature from 4 to 37 degrees C causes a 30-fold increase of Kd for EF-Tu x GTP x Phe-tRNA complexes. The mutation K237E leads to rapid inactivation at the latter temperature. A parallel is found between the order of increasing Kd values for EF-Tus with mutation G316D, A375T and Q124K, respectively, and their order of increasing resistance to kirromycin.     

Interaction of mammalian mitochondrial elongation factor EF-Tu with guanine nucleotides

Elongation factor Tu (EF-Tu) promotes the binding of aminoacyl-tRNA (aa-tRNA) to the acceptor site of the ribosome. During the elongation cycle, EF-Tu interacts with guanine nucleotides, aa-tRNA and its nucleotide exchange factor (EF-Ts). Quantitative determination of the equilibrium dissociation constants that govern the interactions of mammalian mitochondrial EF-Tu (EF-Tu(mt)) with guanine nucleotides was the focus of the work reported here. Equilibrium dialysis with [3H]GDP was used to measure the equilibrium dissociation constant of the EF-Tu(mt) x GDP complex (K(GDP) = 1.0 +/- 0.1 microM). Competition of GTP with a fluorescent derivative of GDP (mantGDP) for binding to EF-Tu(mt) was used to measure the dissociation constant of the EF-Tu(mt) x GTP complex (K(GTP) = 18 +/- 9 microM). The analysis of these data required information on the dissociation constant of the EF-Tu(mt) x mantGDP complex (K(mGDP) = 2.0 +/- 0.5 microM), which was measured by equilibrium dialysis. Both K(GDP) and K(GTP) for EF-Tu(mt) are quite different (about two orders of magnitude higher) than the dissociation constants of the corresponding complexes formed by Escherichia coli EF-Tu. The forward and reverse rate constants for the association and dissociation of the EF-Tu(mt) x GDP complex were determined using the change in the fluorescence of mantGDP upon interaction with EF-Tu(mt). These values are in agreement with a simple equilibrium binding interaction between EF-Tu(mt) and GDP. The results obtained are discussed in terms of the recently described crystal structure of the EF-Tu(mt) x GDP complex.     

Effects of the antibiotic pulvomycin on the elongation factor Tu-dependent reactions. Comparison with other antibiotics

The antibiotic pulvomycin is an inhibitor of protein synthesis that prevents the formation of the ternary complex between elongation factor (EF-) Tu.GTP and aminoacyl-tRNA. In this report, novel aspects of its action on EF-Tu are described. Pulvomycin markedly affects the equilibrium and kinetics of the EF-Tu-nucleotide interaction, particularly of the EF-Tu.GTP complex. The binding affinity of EF-Tu for GTP is increased 1000 times, mainly as the consequence of a dramatic decrease in the dissociation rate of this complex. In contrast, the affinity for GDP is decreased 10-fold due to a marked increase in the dissociation rate of EF-Tu.GDP (25-fold) that mimics the action of EF-Ts, the GDP/GTP exchange factor of EF-Tu. The effects of pulvomycin and EF-Ts can coexist and are simply additive, supporting the conclusion that these two ligands interact with different sites of EF-Tu. This is further confirmed on native PAGE by the ability of EF-Tu to bind the EF-Ts and the antibiotic simultaneously. Pulvomycin enhances the intrinsic EF-Tu GTPase activity, like kirromycin, though to a much more modest extent. As with kirromycin, this stimulation depends on the concentration and nature of the monovalent cations, Li(+) being the most effective one, followed by Na(+), K(+), and NH(4)(+). In the presence of pulvomycin (in contrast to kirromycin), aa-tRNA and/or ribosomes do not enhance the GTPase activity of EF-Tu. The property of pulvomycin to modify selectively the conformation(s) of EF-Tu is also supported by its effect on heat- and urea-dependent denaturation, and tryptic digestion of the protein. Specific differences and similarities between the action of pulvomycin and the other EF-Tu-specific antibiotics are described and discussed.     

Mutant EF-Tu species reveal novel features of the enacyloxin IIa inhibition mechanism on the ribosome

For clarification of the action of a new antibiotic, the analysis of resistant mutants is often indispensable. For enacyloxin IIa we discovered four resistant elongation factor Tu (EF-Tu) species in Escherichia coli with the mutations Q124K, G316D, Q329H, and A375T, respectively. They revealed that enacyloxin IIa sensitivity is dominant in a mixed population of resistant and wild-type EF-Tus. This points to an inhibition mechanism in which EF-Tu is the dominant target of enacyloxin IIa and in which a ribosome with a sensitive EF-Tu blocks mRNA translation for upstream ribosomes with resistant EF-Tus, a mechanism similar to that of the unrelated antibiotic kirromycin. Remarkably, the same mutations are also linked to kirromycin resistance, though the order of their levels of resistance is different from that for enacyloxin IIa. Among the mutant EF-Tus, three different resistance mechanisms can be distinguished: (i) by obstructing enacyloxin IIa binding to EF-Tu. GTP; (ii) by enabling the release of enacyloxin IIa after GTP hydrolysis; and (iii) by reducing the affinity of EF-Tu.GDP. enacyloxin IIa for aminoacyl-tRNA at the ribosomal A-site, which then allows the release of EF-Tu.GDP.enacyloxin IIa. Ala375 seems to contribute directly to enacyloxin IIa binding at the domain 1-3 interface of EF-Tu.GTP, a location that would easily explain the pleiotropic effects of enacyloxin IIa on the functioning of EF-Tu.     

The elongation factor Tu binds aminoacyl-tRNA in the presence of GDP

Escherichia coli elongation factor (EF-Tu) binds aminoacyl-tRNAs (aa-tRNA) not only in the presence of GTP but also in the presence of GDP. Complex formation leads to a protection of the aa-tRNA against nonenzymatic deacylation and digestion by pancreatic ribonuclease, as well as to a protection of EF-Tu against proteolysis by trypsin. The equilibrium constant for the binding of Phe-tRNAPheyeast for example to EF-Tu.GDP has been determined to be 0.7 X 10(5) M-1 which is 2 orders of magnitude lower than the equilibrium constant for Phe-tRNAPheyeast binding to EF-Tu.GTP. In the presence of kirromycin, aminoacyl-tRNA binding to EF-Tu.GDP is not affected as much: Phe-tRNAPheyeast is bound with an equilibrium constant of 3 X 10(5) M-1. While there is also a measurable interaction between EF-Tu.GTP and tRNA, such an interaction cannot be detected with EF-Tu.GDP and tRNA, not even at millimolar concentrations. A so far undetected complex formation between aminoacyl-tRNA and EF-Tu.GTP in the presence of pulvomycin, however, could be detected. The results are discussed in terms of the structural requirements of ternary complex formation and in the light of proofreading schemes involving A-site binding on the E. coli ribosome.     

Isolation and stability of ternary complexes of elongation factor Tu, GTP and aminoacyl-tRNA.

Intact, native EF-Tu, isolated using previously described methods and fully active in binding GTP, was never found to be fully active in binding aminoacyl-tRNA as judged by high performance liquid chromatography (HPLC) gel filtration and zone-interference gel-electrophoresis. In the presence of kirromycin, however, all these EF-Tu.GTP molecules bind aminoacyl-tRNA, although with a drastically reduced affinity. For the first time, the purification of milligram quantities of ternary complexes of EF-Tu.GTP and aminoacyl-tRNA, free of deacylated tRNA and inactive EF-Tu, has become possible using HPLC gel filtration. We also describe an alternative new method for the isolation of the ternary complexes by means of fractional extraction in the presence of polyethylene glycol. In the latter procedure, the solubility characteristics of the ternary complexes are highly reminiscent to those of free tRNA. Concentrated samples of EF-Tu.GMPPNP.aminoacyl-tRNA complexes show a high stability.     

Interaction of a selenocysteine-incorporating tRNA with elongation factor Tu from E.coli.

Selenocysteine-incorporating tRNA(Sec)(UCA), the product of selC, was isolated from E.coli and aminoacylated with serine. The equilibrium dissociation constant for the interaction of Ser-tRNA(Sec)(UCA) with elongation factor Tu.GTP was determined to be 5.0 +/- 2.5 x 10(-8) M. Compared with the dissociation constants of the two elongator Ser-tRNA(Ser) species (Kd = 7 x 10(-10) M), the selenocysteine-incorporating UGA suppressor tRNA has an almost hundred fold weaker affinity for EF-Tu.GTP. This suggests a mechanism by which the Ser-tRNA(Sec) is prevented in recognition of UGA codons. This tRNA is not bound to EF-Tu.GTP and is converted to selenocysteinyl-tRNA(Sec). We also demonstrate the lack of an efficient interaction of Sec-tRNA(Sec)(UCA) with EF-Tu.GTP. The results of this work are in support of a mechanism by which the selenocysteine incorporation at UGA nonsense codons is mediated by an elongation factor other than EF-Tu.GTP.     

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

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 tuf gene 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 EF-Tu.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.     

Transfer of plasmid-borne tuf mutations to the chromosome as a genetic tool for studying the functioning of EF-TuA and EF-TuB in the E. coli cell

The elongation factor EF-Tu of E. coli is a multifunctional protein that lends itself extremely well to studies concerning structure-function relationships. It is encoded by two genes: tufA and tufB. Mutant species of EF-Tu have been obtained by various genetic manipulations, including site- and segment-directed mutagenesis of tuf genes on a vector. The presence of multiple tuf genes in the cell, both chromosomal and plasmid-borne, hampers the characterization of the mutant EF-Tu. We describe a procedure for transferring plasmid-borne tuf gene mutations to the chromosome. Any mutation engineered by genetic manipulation of tuf genes on a vector can be transferred both to the tufA and the tufB position on the chromosome. The procedure facilitated the functional characterization of some of our recently obtained tuf mutations. Of particular relevance is, that it enabled us for the first time to obtain a mutant tufB on the chromosome, encoding an EF-TuB resistant to kirromycin. It thus became possible to study the consequences for growth of tufA inactivation by insertion of bacteriophage Mu. The preliminary evidence obtained suggests that an EF-TuA, active in polypeptide synthesis, is essential for growth whereas such an EF-TuB is dispensable.     

Enacyloxin IIa pinpoints a binding pocket of elongation factor Tu for development of novel antibiotics

Elongation factor (EF-) Tu.GTP is the carrier of aminoacyl-tRNA to the programmed ribosome. Enacyloxin IIa inhibits bacterial protein synthesis by hindering the release of EF-Tu.GDP from the ribosome. The crystal structure of the Escherichia coli EF-Tu.guanylyl iminodiphosphate (GDPNP).enacyloxin IIa complex at 2.3 A resolution presented here reveals the location of the antibiotic at the interface of domains 1 and 3. The binding site overlaps that of kirromycin, an antibiotic with a structure that is unrelated to enacyloxin IIa but that also inhibits EF-Tu.GDP release. As one of the major differences, the enacyloxin IIa tail borders a hydrophobic pocket that is occupied by the longer tail of kirromycin, explaining the higher binding affinity of the latter. EF-Tu.GDPNP.enacyloxin IIa shows a disordered effector region that in the Phe-tRNAPhe.EF-Tu (Thermus aquaticus).GDPNP.enacyloxin IIa complex, solved at 3.1 A resolution, is stabilized by the interaction with tRNA. This work clarifies the structural background of the action of enacyloxin IIa and compares its properties with those of kirromycin, opening new perspectives for structure-guided design of novel antibiotics.     

The influence of different modifications of elongation factor Tu from Escherichia coli on ternary complex formation investigated by fluorescence spectroscopy.

A fluorescence titration assay was used to detect the effects of various modifications of E.coli elongation factor Tu on the formation of the ternary complex with aminoacyl-tRNAs. The treatment of EF-Tu.GDP with TPCK, an analogue of the 3'terminus of aminoacyl-tRNA, was found to have no influence on the conversion of EF-Tu.GDP to 'active' EF-Tu.GTP, but does decrease the affinity of the activated protein for yeast aminoacyl-tRNA by more than three orders of magnitude. Modification of the elongation factor by limited cleavage with trypsin, leading to the excision of amino acid residues 45-58, has only a minor influence on ternary complex formation. The equilibrium dissociation constant of the ternary complex with this trypsin-treated EF-Tu.GTP and E.coli Phe-tRNA(Phe) is only one order of magnitude higher than that of the ternary complex with native EF-Tu. Mutations in the amino acid residues 222 and 375 of EF-Tu also have little effect on ternary complex formation. Compared with TPCK-treated EF-Tu, the affinities of the two mutant species, designated EF-tuAR and EF-TuBO respectively, for [AEDANS-s2C]Tyr-tRNA(Tyr) are only slightly reduced and in the same range as trypsin-cleaved EF-Tu.