Different regions of aminoacyl-tRNA regulate the function of elongation factor Tu

In this work we show that intact aminoacyl-tRNA (aa-tRNA) and its 3' half-molecule, but not its 3' C-C-A-aa fragment, require selective ionic conditions for stimulating the mRNA-independent GTPase of elongation factor Tu (EF-Tu) in the presence of ribosomes.l Stimulation by aa-tRNA and its 3' half-molecule is only observed at 20 and 30 mM Mg2+ and not at 10 mM, where they exert inhibitory activity; by contrast, C-C-A-aa enhances the GTPase activity at all three of these Mg2+ concentrations. Ammonium ion is needed for stimulation by C-C-A-aa, whereas it inhibits the stimulation by aa-tRNA and its 3' half-molecule. The concentration of aminoacylated fragments needed for half-maximum stimulation follows this order: A-Val much greater than C-A-Val greater than C-C-A-Val much greater than 3' Val-tRNA1Val half-molecule greater than Val-tRNA1Val. The extent of maximum stimulation of the EF-Tu GTPase in the presence of ribosomes varies moderately depending on the aa-tRNA species; a clear dependence on the nature of the aminoacyl side chain is observed in the effects of their respective C-C-A-aa fragments tested (C-C-A-Arg, C-C-A-Val, C-C-A-Phe, C-C-A-Met, C-C-A-Lys). In the absence of ribosomes and at low [Mg2+], the one-round GTP hydrolysis by EF-Tu is enhanced by C-C-A-aa fragments, whereas it is inhibited by the corresponding aa-tRNAs. Our results suggest that besides the 3' aminoacylated extremity another region(s) of the aa-tRNA molecule controls the GTPase of EF-Tu. The "unspecific" stimulation by C-C-A-aa and the "specific," aa-tRNA-like effect of the 3' aa-tRNA half-molecule point to the importance of the T chi C loop and stem, as well as of the adjacent regions for the regulation of this function.     

Characterization of the elongation factors from calf brain. 3. Properties of the GTPase activity of EF-1 alpha and mode of action of kirromycin

The GTPase activity of purified EF-1 alpha from calf brain has been studied under various experimental conditions and compared with that of EF-Tu. EF-1 alpha displays a much higher GTPase turnover than EF-Tu in the absence of aminoacyl-tRNA (aa-tRNA) and ribosomes (intrinsic GTPase activity); this is due to the higher exchange rate between bound GDP and free GTP. Also the intrinsic GTPase of EF-1 alpha is enhanced by increasing the concentration of monovalent cations, K+ being more effective than NH+4. Differently from EF-Tu, aa-tRNA is much more active than ribosomes in stimulating the EF-1 alpha GTPase activity. However, ribosomes strongly reinforce the aa-tRNA effect. In the absence of aa-tRNA the rate-limiting step of the GTPase turnover appears to be the hydrolysis of GTP, whereas in its presence the GDP/GTP exchange reaction becomes rate-limiting, since addition of EF-1 beta enhances turnover GTPase activity. Kirromycin moderately inhibits the intrinsic GTPase of EF-1 alpha; this effect turns into stimulation when aa-tRNA is present. Addition of ribosomes abolishes any kirromycin effect. The inability of kirromycin to affect the EF-1 alpha/guanine-nucleotide interaction in the presence of ribosomes shows that, differently from EF-Tu, the EF-1 alpha X GDP/GTP exchange reaction takes place on the ribosome.     

Mutagenesis of three residues, isoleucine-60, threonine-61, and aspartic acid-80, implicated in the GTPase activity of Escherichia coli elongation factor Tu.

The properties of variants of elongation factor (EF) Tu mutated at three positions implicated in its GTPase activity are presented. Mutation I60A, which reduces one wing of a "hydrophobic barrier" screening off the nucleophilic water molecule found at the GTP gamma-phosphate, causes a reduction of the intrinsic GTPase activity contrary to prediction and has practically no influence on other properties. Mutation D80N, which in the isolated G-domain of EF-Tu caused a strong stimulation of the intrinsic GTPase, reduces this activity in the intact molecule. However, whereas for wild-type EF-Tu complex formation with aa-tRNA reduces the GTPase, EF-Tu[D80N] shows a strongly increased activity when bound to Phe-tRNA. Moreover, ribosomes or kirromycin can stimulate its GTPase up to the same level as for wild-type. This indicates that a local destabilization of the magnesium binding network does not per se cause an increased GTPase but does affect its tight regulation. Interestingly, mutant D80N sequestrates EF-Ts by formation of a more stable complex. Substitutions T61A and T61N induce low intrinsic GTPase, and the stimulation by ribosome is less for T61A than for T61N but still detectable, while kirromycin stimulates the GTPase of both mutants equally. This provides more evidence that stimulation by kirromycin and ribosomes follows a different mechanism. The functional implications of these mutations are discussed in the context of a transition state mechanism for catalysis. An alternative structural explanation for the strong conservation of Ile-60 is proposed.     

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)     

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.     

Structural insights into cognate versus near-cognate discrimination during decoding

The structural basis of the tRNA selection process is investigated by cryo-electron microscopy of ribosomes programmed with UGA codons and incubated with ternary complex (TC) containing the near-cognate Trp-tRNA(Trp) in the presence of kirromycin. Going through more than 350 000 images and employing image classification procedures, we find ～8% in which the TC is bound to the ribosome. The reconstructed 3D map provides a means to characterize the arrangement of the near-cognate aa-tRNA with respect to elongation factor Tu (EF-Tu) and the ribosome, as well as the domain movements of the ribosome. One of the interesting findings is that near-cognate tRNA's acceptor stem region is flexible and CCA end becomes disordered. The data bring direct structural insights into the induced-fit mechanism of decoding by the ribosome, as the analysis of the interactions between small and large ribosomal subunit, aa-tRNA and EF-Tu and comparison with the cognate case (UGG codon) offers clues on how the conformational signals conveyed to the GTPase differ in the two cases.     

The antibiotics kirromycin and pulvomycin bind to different sites on the elongation factor Tu from Escherichia coli

Pulvomycin and kirromycin, two antibiotics which inhibit protein biosynthesis in Escherichia coli by complex formation with the elongation factor Tu (EF-Tu), bind to different sites on the protein. While only one molecule of kirromycin can be bound to one molecule of EF-Tu, more than one molecule of pulvomycin interacts with a molecule of EF-Tu. This has been deduced from experiments in which the aminoacyl-tRNA binding and the GTPase activity of EF-Tu were measured in the presence of varying amounts of both antibiotics. These experiments are interpreted to mean that pulvomycin but not kirromycin can replace the other antibiotic in its respective site. Our conclusions are supported by circular dichroism spectroscopy.     

New antibiotic that acts specifically on the GTP-bound form of elongation factor Tu.

The new thiazolyl peptide antibiotic GE2270 A, isolated from Planobispora rosea strain ATCC 53773, is shown to inhibit bacterial protein biosynthesis in vitro by affecting specifically the GTP-bound form of elongation factor Tu (EF-Tu). The 'off' rate of EF-Tu.GTP is slowed down 400-fold, locking GTP on EF-Tu, whereas EF-Tu.GDP is unaffected. Therefore, on the EF-Tu.guanine nucleotide interaction, GE2270 A mimicks the effect of aa-tRNA. In line with this, the binding of aa-tRNA to EF-Tu.GTP is hindered by the antibiotic, as shown by the absence of a stable ternary complex and the inhibition of the enzymatic binding of aa-tRNA to the ribosome. This blocks the elongation cycle. GE2270 A does not essentially modify the intrinsic GTPase activity of EF-Tu, but impairs the stimulation by ribosomes of this reaction. The negative effect of GE2270 A on the EF-Tu.GTP interaction with aa-tRNA bears similarities with that of the structurally unrelated pulvomycin, whereas marked differences were found by comparing the effects of these two antibiotics on EF-Tu.GDP. This work emphasizes the varieties of the transitional conformations which tune the EF-Tu interaction with GTP and GDP.     

Structure-function relationships of elongation factor Tu. Isolation and activity of the guanine-nucleotide-binding domain

The guanine-nucleotide-binding domain (G domain) of elongation factor Tu(EF-Tu) consisting of 203 amino acid residues, corresponding to the N-terminal half of the molecule, has been recently engineered by deleting part of the tufA gene and partially characterized [Parmeggiani, A., Swart, G. W. M., Mortensen, K. K., Jensen, M., Clark, B. F. C., Dente, L. and Cortese, R. (1987) Proc. Natl Acad. Sci. USA 84, 3141-3145]. In an extension of this project we describe here the purification steps leading to the isolation of highly purified G domain in preparative amounts and a number of functional properties. The G domain is a relatively stable protein, though less stable than EF-Tu towards thermal denaturation (t50% = 41.3 degrees C vs. 46 degrees C, respectively). Unlike EF-Tu, its affinity for GDP and GTP, as well as the association and dissociation rates of the relative complexes are similar, as determined under a number of different experimental conditions. Like EF-Tu, the GTPase of the G domain is strongly enhanced by increasing concentrations of Li+, K+, Na+ or NH+4, up to the molar range. The effects of the specific cations shows similarities and diversities when compared to the effects on EF-Tu. K+ and Na+ are the most active followed by NH+4 and Li+ whilst Cs+ is inactive. In the presence of divalent cations, optimum stimulation occurs in the range 3-5 mM, Mg2+ being more effective than Mn2+ and Ca2+. Monovalent and divalent cations are both necessary components for expressing the intrinsic GTPase activity of the G domain. The pH curve of the G domain GTPase displays an optimum at pH 7-8, similar to that of EF-Tu. The 70-S ribosome is the only EF-Tu ligand affecting the G domain in the same manner as that observed with the intact molecule, although the extent of the stimulatory effect is lower. The rate of dissociation of the G domain complexes with GTP and GDP as well as the GTPase activity are also influenced by EF-Ts and kirromycin, but the effects evoked are small and in most cases different from those exerted on EF-Tu. The inability of the G domain to sustain poly(Phe) synthesis is in agreement with the apparent lack of formation of a ternary complex between the G domain.GTP complex and aa-tRNA.(ABSTRACT TRUNCATED AT 400 WORDS)     

The binding of kirromycin to elongation factor Tu. Structural alterations are responsible for the inhibitory action.

The influence of kirromycin on the elongation factor Tu (EF-Tu) in its binary and ternary complexes was investigated. The equilibrium constant for the binding of the antibiotic to EF-Tu . GDP and EF-Tu . GTP was determined by circular dichroism titrations to be 4 x 10(6) M-1, and to EF-Tu . GTP . aa-tRNA by a combination of circular dichroism titrations and hydrolysis protection experiments to be 2 x 10(6) M-1. In the presence of kirromycin the binding of aminoacyl-tRNAs to EF-Tu . GTP is weakened by a factor of two. The antibiotic changes the conformation of the ternary complex in such a way that the aminoacyl moiety of the aminoacyl-tRNA is more accessible to the non-enzymatic hydrolysis. It is concluded that this structural alteration is responsible for the inhibitory action of the antibiotic.     

Aminoacyl-tRNA-elongation factor Tu-ribosome interaction leading to hydrolysis of guanosine 5'-triphosphate

We investigated the elongation factor Tu (EF-Tu) dependent binding of Phe-tRNA and Phe-tRNAs with the nicks at positions 46, 37, and 17 to the Escherichia coli 70S ribosome-poly(U)-tRNAPhe complex. Binding of Phe-tRNA1-45 + 47-76, Phe-tRNA1-36 + 38-76, or Phe-tRNA1-16 + 17-76 to the 70S ribosome has been found to be poly(U) X tRNA dependent and, similar to that of intact Phe-tRNA, is inhibited by the antibiotic thiostrepton. We have further found that, contrary to a previous report [Modolell, J., Cabrer, B., Parmeggiani, A., & Vazquez, D. (1971) Proc. Natl. Acad. Sci. U.S.A. 68, 1796], the EF-Tu-ribosome GTPase mediated by Phe-tRNA is not inhibited by thiostrepton; rather, the drug stimulates the endogenous GTPase of the EF-Tu X 70S ribosome. Phe-tRNA fragments 47-76, 38-76, and 17-76 all promote the EF-Tu X GTPase reaction in the presence of 70S ribosome-poly(U)-tRNAPhe yeast. Moreover, since the GTPase-promoting activities of both the short and long fragments are similar, it appears that the most important aminoacyl transfer ribonucleic acid (aa-tRNA) interaction with EF-Tu occurs alongside its 3' quarter. Thiostrepton slightly stimulates the GTPase activity of these Phe-tRNA fragments. Although the Phe-tRNA1-36 + 38-76 cannot bind to poly(U) during its binding to 70S ribosomes, its binding at high Mg2+ concentration occurs at the A site. Thus, most of the bound modified Phe-tRNA functions as the acceptor in the peptidyltransferase reaction.(ABSTRACT TRUNCATED AT 250 WORDS)     

Substitution of Val20 by Gly in elongation factor Tu. Effects on the interaction with elongation factors Ts, aminoacyl-tRNA and ribosomes

Substitution of V20 by G in the consensus element G18HVDHGK24 of EF-Tu (referred to as EF-TuG20) strongly influences the interaction with GDP as well as the GTPase activity [Jacquet, E. & Parmeggiani, A. (1988) EMBO J. 7, 2861-2867]. In an extension of this work we describe additional properties of the mutated factor, paying particular attention to the interaction with the macromolecular ligands. Our results show that the conformational transitions induced by the mutation strongly favor the regeneration of the active complex EF-TuG20.GTP, almost as effectively as with wild-type EF-Tu in the presence of elongation factor Ts. Addition of elongation factor Ts further enhances the rate of the GDP to GTP exchange of the mutated factor. Remarkably, EF-TuG20.GDP can support the enzymatic binding of aminoacyl-tRNA to ribosome.mRNA at low MgCl2 concentration, an effect that with wild-type EF-Tu can only occur in the presence of kirromycin. Our results show that EF-TuG20.GDP shares common features with the GTP-like conformation induced by kirromycin on wild-type EF-Tu. The ability of the ribosome to activate the EF-TuG20 center for GTP hydrolysis is strongly decreased, while the stimulation by aminoacyl-tRNA is conserved. The ribosomal activity is partially restored by addition of aminoacyl-tRNA plus poly(U), showing that codon/anticodon interaction contribute to correct the anomalous interaction between ternary complex and ribosomes. The impaired activity of EF-TuG20 in poly(Phe) synthesis is related to the degree of defective GTP hydrolysis and, most interestingly, it is characterized by a striking increase of the fidelity of translation at high MgCl2 concentration. This effect probably depends on a more selective recognition of the ternary complex by ribosome.mRNA, as a consequence of a longer pausing of EF-TuG20 on the ribosome. In conclusion, position 20 in EF-Tu is important for coordinating the allosteric mechanisms controlling the action of EF-Tu and its ligands.     

Thermodynamic properties of nucleotide-free EF-Tu from Thermus thermophilus in the presence of low-molecular weight effectors of its GTPase activity

The thermal transition of elongation factor EF-Tu from Thermus thermophilus in the presence of low-molecular weight effectors was studied by differential scanning calorimetry. The effectors of GTPase activity used were the antibiotic kirromycin and the cations Li(+), Na(+), K(+) and NH(4)(+) in the chloride form. The temperature of thermal denaturation and the cooperativity of the transition of nucleotide-free EF-Tu (EF-Tu(f)) in the presence of kirromycin are comparable with those of the EF-Tu x guanosine-5'-[beta,gamma-imido]triphosphate (GppNHp) form, indicating similar conformational states. Increased concentrations of Na(+) and K(+) stabilized EF-Tu(f) in a manner similar to GppNHp. NH(4)(+) decreased the transition temperature of EF-Tu(f) and Li(+) decreased both the temperature and the calorimetric enthalpy of the thermal transition of EF-Tu(f). In the presence of salts, binding of kirromycin had a stabilizing effect on EF-Tu(f). Correlation between the GTPase activity and thermodynamic characteristics of EF-Tu(f) induced by kirromycin in the absence or presence of the cations is discussed.     

The crystal structure of Cys-tRNACys-EF-Tu-GDPNP reveals general and specific features in the ternary complex and in tRNA.

BACKGROUND:. The translation elongation factor EF-Tu in its GTP-bound state forms a ternary complex with any aminoacylated tRNA (aa-tRNA), except initiator tRNA and selenocysteinyl-tRNA. This complex delivers aa-tRNA to the ribosomal A site during the elongation cycle of translation. The crystal structure of the yeast Phe-tRNAPhe ternary complex with Thermus aquaticus EF-Tu-GDPNP (Phe-TC) has previously been determined as one representative of this general yet highly discriminating complex formation. RESULTS: The ternary complex of Escherichia coli Cys-tRNACys and T. aquaticus EF-Tu-GDPNP (Cys-TC) has been solved and refined at 2.6 degrees resolution. Conserved and variable features of the aa-tRNA recognition and binding by EF-Tu-GTP have been revealed by comparison with the Phe-TC structure. New tertiary interactions are observed in the tRNACys structure. A 'kissing complex' is observed in the very close crystal packing arrangement. CONCLUSIONS: The recognition of Cys-tRNACys by EF-Tu-GDPNP is restricted to the aa-tRNA motif previously identified in Phe-TC and consists of the aminoacylated 3' end, the phosphorylated 5' end and one side of the acceptor stem and T stem. The aminoacyl bond is recognized somewhat differently, yet by the same primary motif in EF-Tu, which suggests that EF-Tu adapts to subtle variations in this moiety among all aa-tRNAs. New tertiary interactions revealed by the Cys-tRNACys structure, such as a protonated C16:C59 pyrimidine pair, a G15:G48 'Levitt pair' and an s4U8:A14:A46 base triple add to the generic understanding of tRNA structure from sequence. The structure of the 'kissing complex' shows a quasicontinuous helix with a distinct shape determined by the number of base pairs.     

Mutagenesis of glutamine 290 in Escherichia coli and mitochondrial elongation factor Tu affects interactions with mitochondrial aminoacyl-tRNAs and GTPase activity

Elongation factor Tu (EF-Tu) is responsible for the delivery of the aminoacyl-tRNAs (aa-tRNA) to the ribosome during protein synthesis. The primary sequence of domain II of EF-Tu is highly conserved. However, several residues thought to be important for aa-tRNA binding in this domain are not conserved between the mammalian mitochondrial and bacterial factors. One of these residues is located at position 290 (Escherichia coli numbering). Residue 290 is Gln in most of the prokaryotic factors but is conserved as Leu (L338) in the mammalian mitochondrial factors. This residue is in a loop contacting the switch II region of domain I in the GTP-bound structure. It also helps to form the binding pocket for the 5' end of the aa-tRNA in the ternary complex. In the present work, Leu338 was mutated to Gln (L338Q) in EF-Tu(mt). The complementary mutation was created at the equivalent position in E. coli EF-Tu (Q290L). EF-Tu(mt) L338Q functions as effectively as wild-type EF-Tu(mt) in poly(U)-directed polymerization with both prokaryotic and mitochondrial substrates and in ternary complex formation assays with E. coli aa-tRNA. However, the L338Q mitochondrial variant has a reduced affinity for mitochondrial Phe-tRNA(Phe). E. coli EF-Tu Q290L is more active in poly(U)-directed polymerization with both mitochondrial and prokaryotic substrates and has a higher GTPase activity in both the absence and presence of ribosomes. Surprisingly, while E. coli EF-Tu Q290L is more active in polymerization with mitochondrial Phe-tRNA(Phe), this variant has low activity in the formation of a stable ternary complex with mitochondrial aa-tRNA.     

Thiostrepton inhibition of tRNA delivery to the ribosome

Ribosome-stimulated hydrolysis of guanosine-5'-triphosphate (GTP) by guanosine triphosphatase (GTPase) translation factors drives protein synthesis by the ribosome. Allosteric coupling of GTP hydrolysis by elongation factor Tu (EF-Tu) at the ribosomal GTPase center to messenger RNA (mRNA) codon:aminoacyl-transfer RNA (aa-tRNA) anticodon recognition at the ribosomal decoding site is essential for accurate and rapid aa-tRNA selection. Here we use single-molecule methods to investigate the mechanism of action of the antibiotic thiostrepton and show that the GTPase center of the ribosome has at least two discrete functions during aa-tRNA selection: binding of EF-Tu(GTP) and stimulation of GTP hydrolysis by the factor. We separate these two functions of the GTPase center and assign each to distinct, conserved structural regions of the ribosome. The data provide a specific model for the coupling between the decoding site and the GTPase center during aa-tRNA selection as well as a general mechanistic model for ribosome-stimulated GTP hydrolysis by GTPase translation factors.     

Structure-function relationships in the GTP binding domain of EF-Tu: mutation of Val20, the residue homologous to position 12 in p21.

Val20 of elongation factor Tu (EF-Tu), one of the best-characterized GTP binding proteins, is a variable residue within the consensus motif G-X-X-X-X-G-K involved in the interaction with the phosphates of GDP/GTP. To investigate the structure-function relationships of EF-Tu, which is widely used as a model protein, Val20 has been substituted by Gly using oligonucleotide-directed mutagenesis. The most important effects are: (i) a strong reduction of the intrinsic GTPase activity, (ii) a remarkable enhancement of the association and dissociation rates of EF-TuGly20-GDP, mimicking the effect of elongation factor Ts (EF-Ts) and (iii) the inability of ribosomes to influence the intrinsic GTPase of EF-Tu uncoupled from poly(Phe) synthesis. EF-TuGly20 can sustain poly(Phe) synthesis, albeit at a much lower rate than wild-type EF-TuVal20. As with the latter, poly(Phe) synthesis by EF-TuGly20 is inhibited by the antibiotic kirromycin, but differs remarkably in that it is largely independent of the presence of EF-Ts. According to primary sequence alignment, position 20 is homologous to position 12 of ras protein p21. As in p21, this position in EF-Tu is critical, influencing specifically the GDP/GTP interaction as well as other functions. The effect of the mutation displays diversities but also similarities with the situation reported for p21 having the corresponding residues in position 12. The differences observed with two homologous residues, Gly20 and Gly12 in EF-Tu and p21 respectively, show the importance of a variable residue in a consensus element in defining specific functions of GTP binding proteins.     

Functional-structural analysis of threonine 25, a residue coordinating the nucleotide-bound magnesium in elongation factor Tu

Elongation factor (EF) Tu Thr-25 is a key residue binding the essential magnesium complexed to nucleotide. We have characterized mutations at this position to the related Ser and to Ala, which abolishes the bond to Mg2+, and a double mutation, H22Y/T25S. Nucleotide interaction was moderately destabilized in EF-Tu(T25S) but strongly in EF-Tu(T25A) and EF-Tu(H22Y/T25S). Binding Phe-tRNAPhe to poly(U).ribosome needed a higher magnesium concentration for the latter two mutants but was comparable at 10 mM MgCl2. Whereas EF-Tu(T25S) synthesized poly(Phe), as effectively as wild type, the rate was reduced to 50% for EF-Tu(H22Y/T25S) and was, surprisingly, still 10% for EF-Tu(T25A). In contrast, protection of Phe-tRNAPhe against spontaneous hydrolysis by the latter two mutants was very low. The intrinsic GTPase in EF-Tu(H22Y/T25S) and (T25A) was reduced, and the different responses to ribosomes and kirromycin suggest that stimulation by these two agents follows different mechanisms. Of the mutants, only EF-Tu(T25A) forms a more stable complex with EF-Ts than wild type. This implies that stabilization of the EF-Tu.EF-Ts complex is related to the inability to bind Mg2+, rather than to a decreased nucleotide affinity. These results are discussed in the light of the three-dimensional structure. They emphasize the importance of the Thr-25-Mg2+ bond, although its absence is compatible with protein synthesis and thus with an active overall conformation of EF-Tu.     

Structural basis of the action of pulvomycin and GE2270 A on elongation factor Tu

Pulvomycin inhibits protein synthesis by preventing the formation of the ternary complex between elongation factor Tu (EF-Tu) x GTP and aa-tRNA. In this work, the crystal structure of Thermus thermophilus EF-Tu x pulvomycin in complex with the GTP analogue guanylyl imino diphosphate (GDPNP) at 1.4 A resolution reveals an antibiotic binding site extending from the domain 1-3 interface to domain 2, overlapping the domain 1-2-3 junction. Pulvomycin binding interferes with the binding of the 3'-aminoacyl group, the acceptor stem, and 5' end of tRNA. Only part of pulvomycin overlaps the binding site of GE2270 A, a domain 2-bound antibiotic of a structure unrelated to pulvomycin, which also hinders aa-tRNA binding. The structure of the T. thermophilus EF-Tu x GDPNP x GE2270 A complex at 1.6 A resolution shows that GE2270 A interferes with the binding of the 3'-aminoacyl group and part of the acceptor stem of aa-tRNA but not with the 5' end. Both compounds, pulvomycin more markedly, hinder the correct positioning of domain 1 over domains 2 and 3 that characterizes the active form of EF-Tu, while they affect the domain 1 switch regions that control the EF-Tu x GDP/GTP transitions in different ways. This work reveals how two antibiotics with different structures and binding modes can employ a similar mechanism of action.     

Kirromycin drastically reduces the affinity of Escherichia coli elongation factor Tu for aminoacyl-tRNA

We have studied the interaction between EF-Tu-GDP or EF-Tu-GTP in complex with kirromycin or aurodox (N1-methylkirromycin) and aminoacyl-tRNA, N-acetylaminoacyl-tRNA, or deacylated tRNA. Three independent methods were used: zone-interference gel electrophoresis, GTPase stimulation, and fluorescence. All three methods revealed that kirromycin induces a severe drop in the stability of the complex of EF-Tu-GTP and aminoacyl-tRNA of about 3 orders of magnitude. The affinities of EF-Tu-kirromycin-GTP and EF-Tu-kirromycin-GDP for aa-tRNA were found to be of about the same order of magnitude. We conclude that kirromycin and related compounds do not induce a so-called GTP-like conformation of EF-Tu with respect to tRNA binding. The findings shed new light on the mechanism of action of the antibiotic during the elongation cycle. In contrast to indirect evidence previously obtained in our laboratory [Van Noort et al. (1982) EMBO J. 1, 1199-1205; Van Noort et al. (1986) Proc. Natl. Acad. Sci. U.S.A. 71, 4910-4914], we were unable to demonstrate complexes of EF-Tu-aurodox-GTP/GDP with N-acetylaminoacyl-tRNA or deacylated tRNA by direct detection using zone-interference gel electrophoresis. Modification with N-tosyl-L-phenylalanine chloromethyl ketone (TPCK) decreases the affinity of EF-Tu-kirromycin-GTP for aminoacyl-tRNA, just like it does in the absence of the antibiotic.