Nucleotide binding and GTP hydrolysis by elongation factor Tu from Thermus thermophilus as monitored by proton NMR.

Proton NMR experiments of the GTP/GDP-binding protein EF-Tu from the extremely thermophilic bacterium Thermus thermophilus HB8 in H2O have been performed paying special attention to the resonances in the downfield region (below 10 ppm). Most of these downfield signals are due to hydrogen bonds formed between the protein and the bound nucleotide. However, three downfield resonances appear even in the nucleotide-free EF-Tu. The middle and C-terminal domain (domain II/III) of EF-Tu lacking the GTP/GDP-binding domain gives rise to an NMR spectrum that hints at a well-structured protein. In contrast to native EF-Tu, the domain II/III spectrum contains no resonances in the downfield region. Several downfield resonances can be used as a fingerprint to trace hydrolysis of protein-bound GTP and temperature effects on the EF-Tu.GDP spectra. NMR studies of the binding of guanosine nucleotide analogues (GMPPNP, GMPPCP) to nucleotide-free EF-Tu have been carried out. The downfield resonances of these complexes differ from the spectrum of EF-Tu.GTP. Protected and photolabile caged GTP was bound to EF-Tu, and NMR spectra before and after photolysis were recorded. The progress of the GTP hydrolysis could be monitored using this method. The downfield resonances have been tentatively assigned taking into account the known structural and biochemical aspects of EF-Tu nucleotide-binding site.     

Studies on polypeptide-chain-elongation factors from an extreme thermophile, Thermus thermophilus HB8. 2. Catalytic properties.

Catalytic properties of the elongation factors from Thermus thermophilus HB8 have been studied and compared with those of the factors from Escherichia coli. 1. The formation of a ternary guanine-nucleotide . EF-Tu . EF-Ts complex was demonstrated by gel filtration of the T. thermophilus EF-Tu . EF-Ts complex on a Sephadex G-150 column equilibrated with guanine nucleotide. The occurrence of this type of complex has not yet been proved with the factors from E. coli. 2. The dissociation constants for the complexes of T. thermophilus EF-Tu . EF-Ts with GDP and GTP were 6.1 x 10(-7) M and 1.9 x 10(-6) M respectively. On the other hand, T. thermophilus EF-Tu interacted with GDP and GTP with dissociation constants of 1.1 x 10(-9) M and 5.8 x 10(-8) M respectively. This suggests that the association of EF-Ts with EF-Tu lowered the affinity of EF-Tu for GDP by a factor of about 600 and facilitated the nucleotide exchange reaction. 3. Although the T. thermophilus EF-Tu . EF-Ts complex hardly dissociates into EF-Tu and EF-Ts, a rapid exchange was observed between free EF-Ts and the EF-Tu . EF-Ts complex using 3H-labelled EF-Ts. The exchange reaction was independent on the presence or absence of guanine nucleotides. 4. Based on the above findings, an improved reaction mechanism for the regeneration of EF-Tu . GTP from EF-Tu . GDP is proposed. 5. Studies on the functional interchangeability of EF-Tu and EF-Ts between T. thermophilus and E. coli has revealed that the factors function much more efficiently in the homologous than in the heterologous combination. 6. T. thermophilus EF-Ts could bind E. coli EF-Tu to form an EF-Tu (E. coli) . EF-Ts (T. thermophilus hybrid complex. The complex was found to exist in a dimeric form indicating that the property to form a dimer is attributable to T. thermophilus EF-Ts. On the other hand, no stable complex between E. coli EF-Ts and T. thermophilus EF-Tu has been isolated. 7. The uncoupled GTPase activity of T. thermophilus EF-G was much lower than that of E. coli EF-G. T. thermophilus EF-G formed a relatively stable binary EF-G . GDP complex, which could be isolated on a nitrocellulose membrane filter. The Kd values for EF-G . GDP and EF-G . GTP were 6.7 x 10(-7) M and 1.2 x 10(-5) M respectively. The ternary T. thermophilus EF-G . GDP . ribosome complex was again very stable and could be isolated in the absence of fusidic acid. The stability of the latter complex is probably the cause of the low uncoupled GTPase activity of T. thermophilus EF-G.     

Studies on polypeptide-chain-elongation factors from an extreme thermophile, Thermus thermophilus HB8. 3. Molecular properties.

Molecular properties of the polypeptide chain elongation factors from Thermus thermophilus HB8 have been investigated and compared with those from Escherichia coli. 1. As expected, the factors purified from T. thermophilus were exceedingly heat-stable. Even free EF-Tu not complexed with GDP was stable after heating for 5 min at 60 degrees C. 2. GDP binding activity of T. thermophilus EF-Tu was also stable in various protein denaturants, such as 5.5 M urea, 1.5 M guanidine-HCl, and 4 M LiCl. 3. Amino acid compositions of EF-Tu and EF-G from T. thermophilus were similar to those from E. coli. On the other hand, amino acid composition of T. thermophilus EF-Ts was considerably different from that of E. coli EF-Ts. 4. In contrast to E. coli EF-Tu, T. thermophilus EF-Tu contained no free sulfhydryl group, but one disulfide bond. The disulfide bond was cleaved by sodium borohydride or sodium sulfite under native conditions. The heat stability of the reduced EF-Tu . GDP, as measured by GDP binding activity, did not differ from that of the untreated EF-Tu . GDP. 5. T. thermophilus EF-Ts contained, in addition to one disulfide bond, a sulfhydryl group which could be titrated only after complete denaturation of the protein. 6. Under native conditions one sulfhydryl group of T. thermophilus EF-G was titrated with p-chloromercuribenzoate, while the rate of reaction was very sluggish. The sulfhydryl group appears to be essential for interaction with ribosomes, whereas the ability to form a binary GDP . EF-G complex was not affected by its modification. The protein contained also one disulfide bond. 7. Circular dichroic spectra of EF-Tu from T. thermophilus and E. coli were very similar. Binding of GDP or GTP caused a similar spectral change in both. T. thermophilus and E. coli EF-Tu. On the other hand, the spectra of T. thermophilus EF-G and E. coli EF-G were significantly different, the content of ordered structure being higher in the former as compared to the latter.     

Conformational change of elongation factor Tu (EF-Tu) induced by antibiotic binding. Crystal structure of the complex between EF-Tu.GDP and aurodox

Aurodox is a member of the family of kirromycin antibiotics, which inhibit protein biosynthesis by binding to elongation factor Tu (EF-Tu). We have determined the crystal structure of the 1:1:1 complex of Thermus thermophilus EF-Tu with GDP and aurodox to 2.0-A resolution. During its catalytic cycle, EF-Tu adopts two strikingly different conformations depending on the nucleotide bound: the GDP form and the GTP form. In the present structure, a GTP complex-like conformation of EF-Tu is observed, although GDP is bound to the nucleotide-binding site. This is consistent with previous proposals that aurodox fixes EF-Tu on the ribosome by locking it in its GTP form. Binding of EF-Tu.GDP to aminoacyl-tRNA and mutually exclusive binding of kirromycin and elongation factor Ts to EF-Tu can be explained on the basis of the structure. For many previously observed mutations that provide resistance to kirromycin, it can now be understood how they prevent interaction with the antibiotic. An unexpected feature of the structure is the reorientation of the His-85 side chain toward the nucleotide-binding site. We propose that this residue stabilizes the transition state of GTP hydrolysis, explaining the acceleration of the reaction by kirromycin-type antibiotics.     

Site-directed mutagenesis of the GDP binding domain of bacterial elongation factor Tu

The tertiary structure model of EF-Tu predicts that the amino acid sequence Val-Asp-His-Gly-Lys-Thr-Thr-Leu (residues 20-27) forms a pocket that binds the pyrophosphate group. To test this model we used site-directed mutagenesis to produce forms of EF-Tu altered in this region. The following mutations were constructed: Gly-20, Val-23, Glu-24, Ile-25, and Pro-27. Each protein was labeled with [35S]Met and was tested for its ability to interact with guanosine nucleotides and EF-Ts. The in vivo activity of each altered protein was tested by determining its ability to confer aurodox sensitivity to a resistant host. Mutations at residues 23, 24, 25, and 27 eliminated the ability of EF-Tu to interact with either guanosine nucleotides or EF-Ts in vitro, and these forms were also inactive in vivo. In contrast, the Gly-20 form was nearly as active as wild-type EF-Tu in vitro and in vivo. This mutation is theoretically equivalent to reversion of the Gly to Val transforming mutation of the cellular form of the ras gene product p21, a protein proposed to be structurally similar to EF-Tu in the GDP binding domain. In contrast to its effect in the ras gene, the Val to Gly conversion did not affect the endogenous GTPase of EF-Tu. We conclude that the tertiary structure model is correct in its assignment of the pyrophosphate binding site to residues 23-27; however, there are likely to be some significant differences between the configurations of the GTPases of EF-Tu and p21.     

Functional effects of deleting the coiled-coil motif in Escherichia coli elongation factor Ts.

Elongation factor Ts (EF-Ts) is the guanine nucleotide-exchange factor for elongation factor Tu (EF-Tu) that is responsible for promoting the binding of aminoacyl-tRNA to the mRNA-programmed ribosome. The structure of the Escherichia coli EF-Tu-EF-Ts complex reveals a protruding antiparallel coiled-coil motif in EF-Ts, which is responsible for the dimerization of EF-Ts in the crystal. In this study, the sequence encoding the coiled-coil motif in EF-Ts was deleted from the genome in Escherichia coli by gene replacement. The growth rate of the resulting mutant strain was 70-95% of that of the wild-type strain, depending on the growth conditions used. The mutant strain sensed amino acid starvation and synthesized the nucleotides guanosine 5'-diphosphate 3'-diphosphate and guanosine 5'-triphosphate 3'-diphosphate at a lower cell density than the wild-type strain. Deletion of the coiled-coil motif only partially reduced the ability of EF-Ts to stimulate the guanine nucleotide exchange in EF-Tu. However, the concentration of guanine nucleotides (GDP and GTP) required to dissociate the mutant EF-Tu-EF-Ts complex was at least two orders of magnitude lower than that for the wild-type complex. The results show that the coiled-coil motif plays a significant role in the ability of EF-Ts to compete with guanine nucleotides for the binding to EF-Tu. The present results also indicate that the deletion alters the competition between EF-Ts and kirromycin for the binding to EF-Tu.     

The elongation factor Tu from Escherichia coli, aminoacyl-tRNA, and guanosine tetraphosphate form a ternary complex which is bound by programmed ribosomes

The interaction of the Escherichia coli elongation factor Tu guanosine tetraphosphate complex (EF-Tu ppGpp) with aminoacyl-tRNAs(aa-tRNA) was reinvestigated by gel filtration and hydrolysis protection experiments. These experiments show that EF-Tu X ppGpp like EF-Tu X GDP (Pingoud, A., Block, W., Wittinghofer, A., Wolf, H. & Fischer, E. (1982) J. Biol. Chem. 257, 11261-11267) forms a fairly stable complex with Phe-tRNAPhe, KAss being 0.6 X 10(5) M-1 at 25 degrees C. The binding of the EF-Tu X ppGpp X aa-tRNA complex to programmed ribosomes was investigated by a centrifugation technique. It is shown that this complex is bound codon-specific with KAss = 3 X 10(7) M-1 at 0 degrees C and that it stimulates peptidyl transfer. A numerical estimation of the intracellular concentration of EF-Tu X GTP X aa-tRNA and EF-Tu X ppGpp X aa-tRNA during normal growth and under the stringent response indicates that ppGpp accumulation does affect the EF-Tu X GTP X aa-tRNA concentration but does not lead to major depletion of this pool. Furthermore, due to the higher affinity of EF-Tu X GTP to aa-tRNA and of the ternary complex EF-Tu X GTP X aa-tRNA to the ribosome, EF-Tu X ppGpp X aa-tRNA binding to the ribosome is not significant. According to our measurements and calculations, therefore, a direct participation of EF-Tu in slowing down the rate of protein biosynthesis and improving its accuracy during amino acid starvation is not obvious.     

Hydrogen bond interactions of G proteins with the guanine ring moiety of guanine nucleotides.

We have utilized Raman difference spectroscopy to investigate hydrogen bonding interactions of the guanine moiety in guanine nucleotides with the binding site of two G proteins, EF-Tu (elongation factor Tu from Escherichia coli) and the c-Harvey ras protein, p21 (the gene product of the human c-H-ras proto-oncogene). Raman spectra of proteins complexed with GDP (guanosine 5' diphosphate), IDP (inosine 5' diphosphate), 6-thio-GDP, and 6-18O-GDP were measured, and the various difference spectra were determined. These were compared to the difference spectra obtained in solution, revealing vibrational features of the nucleotide that are altered upon binding. Specifically, we observed significant frequency shifts in the vibrational modes associated with the 6-keto and 2-amino positions of the guanine group of GDP and IDP that result from hydrogen bonding interactions between these groups and the two proteins. These shifts are interpreted as being proportional to the local energy of interaction (delta H) between the two groups and protein residues at the nucleotide binding site. Consistent with the tight binding between the nucleotides and the two proteins, the shifts indicate that the enthalpic interactions are stronger between these two polar groups and protein than with water. In general, the spectral shifts provide a rationale for the stronger binding of GDP and IDP with p21 compared to EF-Tu. Despite the structural similarity of the binding sites of EF-Tu and p21, the strengths of the observed hydrogen bonds at the 6-keto and 2-amino positions vary substantially, by up to a factor of 2.(ABSTRACT TRUNCATED AT 250 WORDS)     

Kinetics and thermodynamics of the interaction of elongation factor Tu with elongation factor Ts, guanine nucleotides, and aminoacyl-tRNA

The exchange of elongation factor Tu (EF-Tu)-bound GTP in the presence and absence of elongation factor Ts (EF-Ts) was monitored by equilibrium exchange kinetic procedures. The kinetics of the exchange reaction were found to be consistent with the formation of a ternary complex EF-Tu X GTP X EF-Ts. The equilibrium association constants of EF-Ts to the EF-Tu X GTP complex and of GTP to EF-Tu X EF-Ts were calculated to be 7 X 10(7) and 2 X 10(6) M-1, respectively. The dissociation rate constant of GTP from the ternary complex was found to be 13 s-1. This is 500 times larger than the GTP dissociation rate constant from the EF-Tu X GTP complex (2.5 X 10(-2) s-1). A procedure based on the observation that EF-Tu X GTP protects the aminoacyl-tRNA molecule from phosphodiesterase I-catalyzed hydrolysis was used to study the interactions of EF-Tu X GTP with Val-tRNAVal and Phe-tRNAPhe. Binding constants of Phe-tRNAPhe and Val-tRNAVal to EF-Tu X GTP of 4.8 X 10(7) and 1.2 X 10(7)M-1, respectively, were obtained. The exchange of bound GDP with GTP in solution in the presence of EF-Ts was also examined. The kinetics of the reaction were found to be consistent with a rapid equilibrium mechanism. It was observed that the exchange of bound GDP with free GTP in the presence of a large excess of the latter was accelerated by the addition of aminoacyl-tRNA. On the basis of these observations, a complete mechanism to explain the interactions among EF-Tu, EF-Ts, guanine nucleotides, and aminoacyl-tRNA has been developed.     

The binding of nucleotides and metal ions to elongation factor Tu from Bacillus stearothermophilus as studied by equilibrium dialysis

EF-Tu from B. stearothermophilus binds divalent metal ions even in the absence of guanine nucleotides. The association constants necessary for characterizing the multiple equilibria between EF-Tu, GDP and the divalent ions magnesium and manganese were determined by equilibrium dialysis. The constants are 4.6 X 10(4) M-1 and 5.4 X 10(5) M-1 for the binding of Mg2 and 1.0 X 10(5) M-1 and 1.1 X 10(6) M-1 for the binding of Mn2 to EF-Tu and EF-Tu . GDP, respectively. In the absence of divalent ions EF-Tu binds GMP, GDP and GTP with association constants of 3 x 10(3) M-1, 1.7 x 10(7) M-1 and 1.3 x 10(6) M-1, respectively. The binding of GDP in the presence of metal ions is an order of magnitude stronger than in the absence of metal ions.     

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.     

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.     

Affinity labeling of the GDP/GTP binding site in Thermus thermophilus elongation factor Tu

Elongation factor Tu from Thermus thermophilus was treated successively with periodate-oxidized GDP or GTP and cyanoborohydride. Covalently modified cyanogen bromide or trypsin fragments of the protein were isolated, and the position of their modification was determined. Lysine residues 52 and 137 were heavily labeled, lysine-137 being considerably more reactive in the GTP form as compared to the GDP form of the protein. These residues are in the proximity of the GDP/GTP binding site. Lys-325 was also labeled, but to a lower extent. The part of the EF-Tu containing residue 52 is missing in crystallized EF-Tu.GDP from Escherichia coli [Jurnak, F. (1985) Science (Washington, D.C.) 230, 32-36]. These results place the part of T. thermophilus EF-Tu corresponding to the missing fragment in E. coli EF-Tu in the vicinity of the nucleotide binding site and allow its role in the interaction with aminoacyl-tRNA and elongation factor Ts to be evaluated. Cross-linking of EF-Tu.GDP by irradiation at 257 nm showed that a sequence of 10 amino acids residues which is found in the Thermus thermophilus elongation factor Tu but not in other homologous bacterial proteins is located in the vicinity of the GDP/GTP binding site.     

Purification and characterization of Saccharomyces cerevisiae mitochondrial elongation factor Tu

Yeast mitochondrial elongation factor Tu (EF-Tu) was purified 200-fold from a mitochondrial extract of Saccharomyces cerevisiae to yield a single polypeptide of Mr = approximately 47,000. The factor was detected by complementation with Escherichia coli elongation factor G and ribosomes in an in vitro phenylalanine polymerization reaction. Mitochondrial EF-Tu, like E. coli EF-Tu, catalyzes the binding of aminoacyl-tRNA to ribosomes and possesses an intrinsic GTP hydrolyzing activity which can be activated either by kirromycin or by ribosomes. Kinetic and binding analyses of the interactions of mitochondrial EF-Tu with guanine nucleotides yielded affinity constants for GTP and GDP of approximately 5 and 25 microM, respectively. The corresponding affinity constants for the E. coli factor are approximately 0.3 and 0.003 microM, respectively. In keeping with these observations, we found that purified mitochondrial EF-Tu, unlike E. coli EF-Tu, does not contain endogenously bound nucleotide and is not stabilized by GDP. In addition, we have been unable to detect a functional counterpart to E. coli EF-Ts in extracts of yeast mitochondria and E. coli EF-Ts did not detectably stimulate amino acid polymerization with mitochondrial EF-Tu or enhance the binding of guanine nucleotides to the factor. We conclude that while yeast mitochondrial EF-Tu is functionally analogous to and interchangeable with E. coli EF-Tu, its affinity for guanine nucleotides and interaction with EF-Ts are quite different from those of E. coli EF-Tu.     

Identification of the N-tosyl-L-phenylalanyl chloromethylketone modification site in Thermus thermophilus elongation factor Tu

EF-Tu from Thermus thermophilus was first labelled with N-[14C]tosyl-L-phenylalaninechloromethylketone and then cleaved by the combined action of CNBr and trypsin. The resulting peptides were separated by reversed-phase HPLC. Analysis of the isolated, labelled peptide led to the identification of a sequence which was identical to residues 76-88 in T. thermophilus EF-Tu. The TPCK reactive site is at Cys-82. Kinetic measurements of the incorporation of TPCK into native EF-Tu and EF-Tu nicked at position Arg-59 were performed. The results provide evidence that the cleavage of the peptide bond between Arg-59 and Gly-60 does not lead to a dramatic conformational change of EF-Tu at the aa-tRNA binding site.     

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.     

Sequence and identification of the nucleotide binding site for the elongation factor Tu from Thermus thermophilus HB8.

Two structural genes for the Thermus thermophilus elongation factor Tu (tuf) were identified by cross-hybridization with the tufA gene from E. coli. The sequence of one of these tuf genes, localized on a 6.6 kb Bam HI fragment, was determined and confirmed by partial protein sequencing of an authentic elongation factor Tu from T. thermophilus HB8. Expression of this tuf gene in E. coli minicells provided a low amount of immuno-precipitable thermophilic EF-Tu. Affinity labeling of the T. thermophilus EF-Tu and sequence comparison with homologous proteins from other organisms were used to identify the guanosine-nucleotide binding domain.     

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.     

Identification of thermodynamically relevant interactions between EF-Tu and backbone elements of tRNA

A set of 45 different tRNAs, each containing a single deoxynucleotide substitution covering the upper half of the molecule was used in conjunction with a high-throughput ribonuclease protection assay to investigate the thermodynamic role of 2' hydroxyl groups in stabilizing a complex with elongation factor Tu (EF-Tu) from Thermus thermophilus. Five distinct 2' hydroxyl groups were identified where substitution with a proton resulted in an approximately tenfold decrease in the binding affinity. The same five 2' hydroxyl groups reduced the affinity of the interaction with the nearly identical Thermus aquaticus EF-Tu. Four of these 2' hydroxyl groups were observed to form hydrogen bonds in a co-crystal structure of tRNA(Phe) and T. aquaticus EF-Tu, while the fifth 2' hydroxyl group can be associated with an intramolecular hydrogen bond in the tRNA. However, four additional hydrogen bonds to 2' hydroxyl groups observed in the crystal structure show no thermodynamic effect upon disruption. Some of these discrepancies may be reconciled based on the unbound structures of the protein and RNA.