Effects of nucleotide- and aurodox-induced changes in elongation factor Tu conformation upon its interactions with aminoacyl transfer RNA. A fluorescence study

The effects of GDP and of aurodox (N-methylkirromycin) on the affinity of elongation factor Tu (EF-Tu) for aminoacyl-tRNA (aa-tRNA) have been quantified spectroscopically by using Phe-tRNA(Phe)-Fl8, a functionally active analogue of Phe-tRNA(Phe) with a fluorescein dye convalently attached to the s4U-8 base. The association of EF-Tu.GDP with Phe-tRNA(Phe)-Fl8 resulted in an average increase of 33% in fluorescein emission intensity. This spectral change was used to monitor the extent of ternary complex formation as a function of EF-Tu.GDP concentration, and hence to obtain a dissociation constant, directly and at equilibrium, for the EF-Tu.GDP-containing ternary complex. The Kd for the Phe-tRNA(Phe)-Fl8.EF-Tu.GDP complex was found to average 28.5 microM, more than 33,000-fold greater than the Kd of the Phe-tRNA(Phe)-Fl8.EF-Tu.GTP complex under the same conditions. In terms of free energy, the delta G degree for ternary complex formation at 6 degrees C was -11.5 kcal/mol with GTP and -5.8 kcal/mol with GDP. Thus, the hydrolysis of the ternary complex GTP results in a dramatic decrease in the affinity of EF-Tu for aa-tRNA, thereby facilitating the release of EF-Tu.GDP from the aa-tRNA on the ribosome. Aurodox (200 microM) decreased the Kd of the GDP complex by nearly 20-fold, to 1.46 microM, and increased the Kd of the GTP complex by at least 6-fold. The binding of aurodox to EF-Tu therefore both considerably strengthens EF-Tu.GDP affinity for aa-tRNA and also weakens EF-Tu.GTP affinity for aa-tRNA.(ABSTRACT TRUNCATED AT 250 WORDS)     

Direct determination of the association constant between elongation factor Tu X GTP and aminoacyl-tRNA using fluorescence

We have investigated the formation of the aa-tRNA X EF-Tu X GTP ternary complex spectroscopically by monitoring a fluorescence change that accompanies the association of EF-Tu X GTP with Phe-tRNAPhe-F8, a functionally active analogue of Phe-tRNAPhe with a fluorescein moiety covalently attached to the s4U-8 base. With this approach, the protein-nucleic acid interaction could be examined by direct means and at equilibrium. The fluorescence emission intensity of each Phe-tRNAPhe-F8 increased by 36-55% upon association with EF-Tu X GTP, depending on the solvent conditions. Thus, when Phe-tRNAPhe-F8 was titrated with EF-Tu X GTP, the extent of ternary complex formation was determined from the increase in emission intensity. A nonlinear least-squares analysis of the titration data yielded a dissociation constant of 0.85 nM for the ternary complex in 50 mM N-(2-hydroxyethyl)piperazine-N'-2-ethanesulfonic acid (pH 7.6), 10 mM MgCl2, and 50 mM NH4Cl, at 6 degrees C. The delta H degrees of this interaction, determined by the temperature dependence of Kd, was -16 kcal/mol; the delta S degrees was therefore -16 cal mol-1 deg-1 at 6 degrees C in this buffer. In a more physiological polycation-containing solvent ("polymix"), the Kd was 4.7 nM. The ionic strength dependence of ternary complex formation showed that a minimum of two salt bridges and a substantial nonelectrostatic contribution are involved in the binding of aa-tRNA to EF-Tu. The affinities of unmodified aa-tRNAs for EF-Tu X GTP were determined by their abilities to compete with the fluorescent aa-tRNA for binding to the protein.(ABSTRACT TRUNCATED AT 250 WORDS)     

Enacyloxin IIa, an inhibitor of protein biosynthesis that acts on elongation factor Tu and the ribosome.

This work analyzes the action of enacyloxin Ila, an inhibitor of bacterial protein biosynthesis. Enacyloxin IIa [IC50 on poly(Phe) synthesis approximately 70 nM] is shown to affect the interaction between elongation factor (EF) Tu and GTP or GDP; in particular, the dissociation of EF-Tu-GTP is strongly retarded, causing the Kd of EF- Tu-GTP to decrease from 500 to 0.7 nM. In its presence, the migration velocity of both GTP- and GDP-bound EF-Tu on native PAGE is increased. The stimulation of EF-Tu-GDP dissociation by EF-Ts is inhibited. EF- Tu-GTP can still form a stable complex with aminoacyl-tRNA (aa-tRNA), but it no longer protects aa-tRNA against spontaneous deacylation, showing that the EF-Tu-GTP orientation with respect to the 3' end of aa-tRNA is modified. However, the EF-Tu-dependent binding of aa-tRNA to the ribosomal A-site is impaired only slightly by the antibiotic and the activity of the peptidyl-transferase center, as determined by puromycin reactivity, is not affected. In contrast, the C-terminal incorporation of Phe into poly(Phe)-tRNA bound to the P-site is inhibited, an effect that is observed if Phe-tRNA is bound to the A-site nonenzymatically as well. Thus, enacyloxin IIa can affect both EF-Tu and the ribosomal A-site directly, inducing an anomalous positioning of aa-tRNA, that inhibits the incorporation of the amino acid into the polypeptide chain. Therefore, it is the first antibiotic found to have a dual specificity targeted to EF-Tu and the ribosome.     

Interaction of mitochondrial elongation factor Tu with aminoacyl-tRNA and elongation factor Ts

Elongation factor (EF) Tu promotes the binding of aminoacyl-tRNA (aa-tRNA) to the acceptor site of the ribosome. This process requires the formation of a ternary complex (EF-Tu.GTP.aa-tRNA). EF-Tu is released from the ribosome as an EF-Tu.GDP complex. Exchange of GDP for GTP is carried out through the formation of a complex with EF-Ts (EF-Tu.Ts). Mammalian mitochondrial EF-Tu (EF-Tu(mt)) differs from the corresponding prokaryotic factors in having a much lower affinity for guanine nucleotides. To further understand the EF-Tu(mt) subcycle, the dissociation constants for the release of aa-tRNA from the ternary complex (K(tRNA)) and for the dissociation of the EF-Tu.Ts(mt) complex (K(Ts)) were investigated. The equilibrium dissociation constant for the ternary complex was 18 +/- 4 nm, which is close to that observed in the prokaryotic system. The kinetic dissociation rate constant for the ternary complex was 7.3 x 10(-)(4) s(-)(1), which is essentially equivalent to that observed for the ternary complex in Escherichia coli. The binding of EF-Tu(mt) to EF-Ts(mt) is mutually exclusive with the formation of the ternary complex. K(Ts) was determined by quantifying the effects of increasing concentrations of EF-Ts(mt) on the amount of ternary complex formed with EF-Tu(mt). The value obtained for K(Ts) (5.5 +/- 1.3 nm) is comparable to the value of K(tRNA).     

Directed mutagenesis identifies amino acid residues involved in elongation factor Tu binding to yeast Phe-tRNAPhe

The co-crystal structure of Thermus aquaticus elongation factor Tu.guanosine 5'- [beta,gamma-imido]triphosphate (EF-Tu.GDPNP) bound to yeast Phe-tRNA(Phe) reveals that EF-Tu interacts with the tRNA body primarily through contacts with the phosphodiester backbone. Twenty amino acids in the tRNA binding cleft of Thermus Thermophilus EF-Tu were each mutated to structurally conservative alternatives and the affinities of the mutant proteins to yeast Phe-tRNA(Phe) determined. Eleven of the 20 mutations reduced the binding affinity from fourfold to >100-fold, while the remaining ten had no effect. The thermodynamically important residues were spread over the entire tRNA binding interface, but were concentrated in the region which contacts the tRNA T-stem. Most of the data could be reconciled by considering the crystal structures of both free EF-Tu.GTP and the ternary complex and allowing for small (1.0 A) movements in the amino acid side-chains. Thus, despite the non-physiological crystallization conditions and crystal lattice interactions, the crystal structures reflect the biochemically relevant interaction in solution.     

Dynamics of Recognition between tRNA and elongation factor Tu

Elongation factor Tu (EF-Tu) binds to all standard aminoacyl transfer RNAs (aa-tRNAs) and transports them to the ribosome while protecting the ester linkage between the tRNA and its cognate amino acid. We use molecular dynamics simulations to investigate the dynamics of the EF-Tu·guanosine 5′-triphosphate·aa-tRNA^Cys complex and the roles played by Mg²⁺ ions and modified nucleosides on the free energy of protein·RNA binding. Individual modified nucleosides have pronounced effects on the structural dynamics of tRNA and the EF-Tu·Cys-tRNA^Cys interface. Combined energetic and evolutionary analyses identify the coevolution of residues in EF-Tu and aa-tRNAs at the binding interface. Highly conserved EF-Tu residues are responsible for both attracting aa-tRNAs as well as providing nearby nonbonded repulsive energies that help fine-tune molecular attraction at the binding interface. In addition to the 3′ CCA end, highly conserved tRNA nucleotides G1, G52, G53, and U54 contribute significantly to EF-Tu binding energies. Modification of U54 to thymine affects the structure of the tRNA common loop resulting in a change in binding interface contacts. In addition, other nucleotides, conserved within certain tRNA specificities, may be responsible for tuning aa-tRNA binding to EF-Tu. The trend in EF-Tu·Cys-tRNA^Cys binding energies observed as the result of mutating the tRNA agrees with experimental observation. We also predict variations in binding free energies upon misacylation of tRNA^Cys with d-cysteine or O-phosphoserine and upon changing the protonation state of l-cysteine. Principal components analysis in each case reveals changes in the communication network across the protein·tRNA interface and is the basis for the entropy calculations.     

The ternary complex of aminoacylated tRNA and EF-Tu-GTP. Recognition of a bond and a fold

The refined crystal structure of the ternary complex of yeast Phe-tRNA^Phe, Thermus aquaticus elongation factor EF-Tu and the non-hydrolyzable GTP analog, GDPNP, revelas many details of the EF-Tu recognition of aminoacylated tRNA (aa-tRNA). EF-Tu-GTP recognizes the aminoacyl bond and one side of the backbone fold of the acceptor helix and has a high affinity for all ordinary elongator aa-tRNAs by binding to this aa-tRNA motif. Yet, the binding of deacylated tRNA, initiator tRNA, and selenocysteine-specific tRNA (tRNA^Sec) is effectively discriminated against. Subtle rearrangements of the binding pocket may occur to optimize the fit to any side chain of the aminoacyl group and interactions with EF-Tu stabilize the 3′-aminoacyl isomer of aa-tRNA. A general complementarity is observed in the location of the binding sites in tRNA for synthetases and for EF-Tu. The complex formation is highly specific for the GTP-bound conformation of EF-Tu, which can explain the effects of various mutants.     

Interaction of methionine-specific tRNAs from Escherichia coli with immobilized elongation factor Tu

The interaction of three different Met-tRNAsMet from E. coli with bacterial elongation factor (EF) Tu X GTP was investigated by affinity chromatography. Met-tRNAfMet which lacks the base pair at the end of the acceptor stem binds only weakly to EF-Tu X GTP, while Met-tRNAmMet has a high affinity for the elongation factor. A modified Met-tRNAfMet which has a C1-G72 base pair binds much more strongly to immobilized EF-Tu X GTP than the native aminoacyl(aa)-tRNA with non-base-paired C1A72 at this position, demonstrating that the base pair including the first nucleotide in the tRNA is one of the essential structural requirements for the aa-tRNA X EF-Tu X GTP ternary complex formation.     

Conformational changes of aminoacyl-tRNA and uncharged tRNA upon complex formation with polypeptide chain elongation factor Tu

The conformation change of Thermus thermophilus tRNA(1Ile) upon complex formation with T. thermophilus elongation factor Tu (EF-Tu) was studied by analysis of the circular dichroism (CD) bands at 315 nm (due to the 2-thioribothymidine residue in the T-loop) and at 295 nm (due to the core structure of tRNA). Formation of the ternary complex of isoleucyl-tRNA(1Ile) and EF-Tu.GTP increased the intensities of these CD bands, indicating stabilization of the association between the T-loop and the D-loop and also a significant conformation change of the core region. Upon complex formation of EF-Tu.GTP and uncharged tRNA, however, the conformation of the core region is not changed, while the association of the two loops is still stabilized. On the other hand, the binding with EF-Tu.GDP does not appreciably affect the conformation of isoleucyl-tRNA or uncharged tRNA. These indicate the importance of the gamma-phosphate group of GTP and the aminoacyl group in the formation of the active complex of aminoacyl-tRNA and EF-Tu.GTP.     

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.     

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.     

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.     

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)     

tRNA and the guanosinetriphosphatase activity of elongation factor Tu

Different sites of the tRNA molecule influence the activity of the elongation factor Tu (EF-Tu) center for GTP hydrolysis [Parlato, G., Pizzano, R., Picone, D., Guesnet, J., Fasano, O., & Parmeggiani, A. (1983) J. Biol. Chem. 258, 995-1000]. Continuing these studies, we have investigated some aspects of (a) the effect of different tRNA(Phe) species, including Ac-Phe-tRNA(Phe) and 3'-truncated tRNA-CCA in the presence and absence of codon-anticodon interaction, and (b) the effect of occupation of the ribosomal P-site by different tRNA(Phe) species. Surprisingly, we have found that 3'-truncated tRNA can enhance the GTPase activity in the presence of poly(U), in contrast to its inhibitory effect in the absence of codon-anticodon interaction. Moreover, Ac-Phe-tRNA(Phe) was found to have some stimulatory effect on the ribosome EF-Tu GTPase in the presence of poly(U). These results indicate that under specific conditions the 3'-terminal end and a free terminal alpha-NH2 group are not essential for the stimulation of the catalytic center of EF-Tu; therefore, the same structure of the tRNA molecule can act as a stimulator or an inhibitor of EF-Tu functions, depending on the presence of codon-anticodon interaction and on the concentration of monovalent and divalent cations. EF-Tu-GTP does not recognize a free ribosomal P-site from a P-site occupied by the different tRNA(Phe) species. When EF-Tu acts as a component of the ternary complex formed with GTP and aa-tRNA, the presence of tRNA in the P-site strongly increases the GTPase activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

The reaction of ribosomes with elongation factor Tu.GTP complexes. Aminoacyl-tRNA-independent reactions in the elongation cycle determine the accuracy of protein synthesis

The fidelity of protein synthesis depends on the rate constants for the reaction of ribosomes with ternary complexes of elongation factor Tu (EF-Tu), GTP, and aminoacyl (aa)-tRNA. By measuring the rate constants for the reaction of poly(U)-programmed ribosomes with a binary complex of elongation factor (EF-Tu) and GTP we have shown that two of the key rate constants in the former reaction are determined exclusively by ribosome-EF-Tu interactions and are not affected by the aa-tRNA. These are the rate constant for GTP hydrolysis, which plays an important role in the fidelity of ternary complex selection by the ribosome, and the rate constant for EF-Tu.GDP dissociation from the ribosome, which plays an equally important role in subsequent proofreading of the aa-tRNA. We conclude that the fidelities of ternary complex selection and proofreading are fundamentally dependent on ribosome-EF-Tu interactions. These interactions determine the absolute value of the rate constants for GTP hydrolysis and EF-Tu.GDP dissociation. The ribosome then uses these rate constants as internal standards to measure, respectively, the rate constants for ternary complex and aa-tRNA dissociation from the ribosome. These rates, in turn, are highly dependent on whether the ternary complex and aa-tRNA are cognate or near-cognate to the codon being translated.     

Ribosome interactions of aminoacyl-tRNA and elongation factor Tu in the codon-recognition complex

The mRNA codon in the ribosomal A-site is recognized by aminoacyl-tRNA (aa-tRNA) in a ternary complex with elongation factor Tu (EF-Tu) and GTP. Here we report the 13 A resolution three-dimensional reconstruction determined by cryo-electron microscopy of the kirromycin-stalled codon-recognition complex. The structure of the ternary complex is distorted by binding of the tRNA anticodon arm in the decoding center. The aa-tRNA interacts with 16S rRNA, helix 69 of 23S rRNA and proteins S12 and L11, while the sarcin-ricin loop of 23S rRNA contacts domain 1 of EF-Tu near the nucleotide-binding pocket. These results provide a detailed snapshot view of an important functional state of the ribosome and suggest mechanisms of decoding and GTPase activation.     

Mechanism of the chemical step for the guanosine triphosphate (GTP) hydrolysis catalyzed by elongation factor Tu

Elongation factor Tu (EF-Tu), the protein responsible for delivering aminoacyl-tRNAs (aa-tRNAs) to ribosomal A site during translation, belongs to the group of guanosine-nucleotide (GTP/GDP) binding proteins. Its active 'on'-state corresponds to the GTP-bound form, while the inactive 'off'-state corresponds to the GDP-bound form. In this work we focus on the chemical step, GTP+H(2)O-->GDP+Pi, of the hydrolysis mechanism. We apply molecular modeling tools including molecular dynamics simulations and the combined quantum mechanical-molecular mechanical calculations for estimates of reaction energy profiles for two possible arrangements of switch II regions of EF-Tu. In the first case we presumably mimic binding of the ternary complex EF-Tu.GTP.aa-tRNA to the ribosome and allow the histidine (His85) side chain of the protein to approach the reaction active site. In the second case, corresponding to the GTP hydrolysis by EF-Tu alone, the side chain of His85 stays away from the active site, and the chemical reaction GTP+H(2)O-->GDP+Pi proceeds without participation of the histidine but through water molecules. In agreement with the experimental observations which distinguish rate constants for the fast chemical reaction in EF-Tu.GTP.aa-tRNA.ribosome and the slow spontaneous GTP hydrolysis in EF-Tu, we show that the activation energy barrier for the first scenario is considerably lower compared to that of the second case.     

Structural features in aminoacyl-tRNAs required for recognition by elongation factor Tu

In bacterial polypeptide synthesis aminoacyl-tRNA (aa-tRNA) bound to elongation factor Tu (EF-Tu) and GTP is part of a crucial intermediate ribonucleoprotein complex involved in the decoding of messenger RNA. The conformation and topology as well as the affinity of the macromolecules in this ternary aa-tRNA X EF-Tu X GTP complex are of fundamental importance for the nature of the interaction of the complex with the ribosome. The structural elements of aa-tRNA required for interaction with EF-Tu and GTP and the resulting functional implications are presented here.     

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.