Wild-type Elongation Factor Tu Research Articles

Design and properties of efficient tRNA:EF-Tu FRET system for studies of ribosomal translation.

Formation of the ternary complex between GTP-bound form of elongation factor Tu (EF-Tu) and aminoacylated transfer RNA (aa-tRNA) is a key event in protein biosynthesis. Here we show that fluorescently modified Escherichia coli EF-Tu carrying three mutations, C137A, C255V and E348C, and fluorescently modified Phe-tRNA(Phe) form functionally active ternary complex that has properties similar to those of the naturally occurring (unmodified) complex. Similarities include the binding and binding rate constants, behavior in gel retardation assay, as well as activities in tRNA protection and in vitro translation assays. Proper labeling of EF-Tu was demonstrated in MALDI mass spectroscopy experiments. To generate the mutant EF-Tu, a series of genetic constructions were performed. Two native cysteine residues in the wild-type EF-Tu at positions 137 and 255 were replaced by Ala and Val, respectively, and an additional cysteine was introduced either in position 324 or 348. The assembly FRET assay showed a 5- to 7-fold increase of Cy5-labeled EF-Tu E348C mutant fluorescence upon formation of ternary complex with charged tRNA(Phe)(Cy3-labeled) when the complex was excited at 532 nm and monitored at 665 nm. In a control experiment, we did not observe FRET using uncharged tRNA(Phe)(Cy3), nor with wild-type EF-Tu preparation that was allowed to react with Cy5 maleimide, nor in the absence of GTP. The results obtained demonstrate that the EF-Tu:tRNA FRET system described can be used for investigations of ribosomal translation in many types of experiments.

Elongation Factor Tu Mutants Expand Amino Acid Tolerance of Protein Biosynthesis System

Nonnatural amino acids have been introduced into proteins using expanded protein biosynthesis systems. However, some nonnatural amino acids, especially those containing large aromatic groups, are not efficiently incorporated into proteins. Reduced binding efficiency of aminoacylated tRNAs to elongation factor Tu (EF-Tu) is likely to limit incorporation of large amino acids. Our previous studies suggested that tRNAs carrying large nonnatural amino acids are bound less tightly to EF-Tu than natural amino acids. To expand the availability of nonnatural mutagenesis, EF-Tu from the E. coli translation system was improved to accept such large amino acids. We synthesized EF-Tu mutants, in which the binding pocket of the aminoacyl moiety of aminoacyl-tRNA was enlarged. L-1-Pyrenylalanine, L-2-pyrenylalanine, and DL-2-anthraquinonylalanine, which are hardly or only slightly incorporated with the wild-type EF-Tu, were successfully incorporated into a protein using these EF-Tu mutants.

Effects of mutagenesis of Gln97 in the switch II region of Escherichia coli elongation factor Tu on its interaction with guanine nucleotides, elongation factor Ts, and aminoacyl-tRNA.

Elongation factor Tu (EF-Tu) delivers aminoacyl-tRNA to the A-site of the ribosome. In a multiple-sequence alignment of prokaryotic EF-Tu's, Gln97 is nearly 100% conserved. In contrast, in mammalian mitochondrial EF-Tu's, the corresponding position is occupied by a conserved proline residue. Gln97 is located in the switch II region in the GDP/GTP binding domain of EF-Tu. This domain undergoes a significant structural rearrangement upon GDP/GTP exchange. To investigate the role of Gln97 in bacterial EF-Tu, the E. coli EF-Tu variant Q97P was prepared. The Q97P variant displayed no activity in the incorporation of [(14)C]Phe on poly(U)-programmed E. coli ribosomes. The Q97P variant bound GDP more tightly than the wild-type EF-Tu with K(d) values of 7.5 and 12 nM, respectively. The intrinsic rate of GDP exchange was 2-3-fold lower for the Q97P variant than for wild-type EF-Tu in the absence of elongation factor Ts (EF-Ts). Addition of EF-Ts equalized the GDP exchange rate between the variant and wild-type EF-Tu. The variant bound GTP at 3-fold lower levels than the wild-type EF-Tu. Strikingly, the Q97P variant was completely inactive in ternary complex formation, accounting for its inability to function in polymerization. The structural basis of these observations is discussed.

Essential Role of Histidine 84 in Elongation Factor Tu for the Chemical Step of GTP Hydrolysis on the Ribosome

Elongation factor Tu (EF-Tu) is a GTP-binding protein that delivers aminoacyl-tRNA to the A site of the ribosome during protein synthesis. The mechanism of GTP hydrolysis in EF-Tu on the ribosome is poorly understood. It is known that mutations of a conserved histidine residue in the switch II region of the factor, His84 in Escherichia coli EF-Tu, impair GTP hydrolysis. However, the partial reaction which is directly affected by mutations of His84 was not identified and the effect on GTP hydrolysis was not quantified. Here, we show that the replacement of His84 with Ala reduces the rate constant of GTP hydrolysis more than 106-fold, whereas the preceding steps of ternary complex binding to the ribosome, codon recognition and, most importantly, the GTPase activation step are affected only slightly. These results show that His84 plays a key role in the chemical step of GTP hydrolysis. Rate constants of GTP hydrolysis by wild-type EF-Tu, measured using the slowly hydrolyzable GTP analog, GTPγS, showed no dependence on pH, indicating that His84 does not act as a general base. We propose that the catalytic role of His84 is to stabilize the transition state of GTP hydrolysis by hydrogen bonding to the attacking water molecule or, possibly, the γ-phosphate group of GTP.

GE2270A-resistant Mutations in Elongation Factor Tu Allow Productive Aminoacyl-tRNA Binding to EF-Tu·GTP·GE2270A Complexes

The antibiotic GE2270A prevents stable complex formation between elongation factor Tu (EF-Tu) and aminoacyl-tRNA (aatRNA). In Escherichia coli we characterized two mutant EF-Tu species with either G257S or G275A that lead to high GE2270A resistance in poly(Phe) synthesis, which at least partially explains the high resistance of EF-Tu1 from GE2270A producer Planobispora rosea to its own antibiotic. Both E.coli mutants were unexpectedly found to bind GE2270A nearly as well as wild-type (wt) EF-Tu in their GTP-bound conformations. Both G257S and G275A are in or near the binding site for the 3′ end of aatRNA. The G257S mutation causes a 2.5-fold increase in affinity for aatRNA, whereas G275A causes a 40-fold decrease. In the presence of GE2270A, wt EF-Tu shows a drop in aatRNA affinity of at least four orders of magnitude. EF-Tu[G275S] and EF-Tu[G275A] curtail this drop to about two or one order, respectively. It thus appears that the resistance mutations do not prevent GE2270A from binding to EF-Tu·GTP and that the mutant EF-Tus may accommodate GE2270A and aatRNA simultaneously. Interestingly, in their GDP-bound conformations the mutant EF-Tus have much less affinity for GE2270A than wt EF-Tu. The latter is explained by a recent crystal structure of the EF-Tu·GDP·GE2270A complex, which predicts direct steric problems between GE2270A and the mutated G257S or G275A. These mutations may cause a dislocation of GE2270A in complex with GTP-bound EF-Tu, which then no longer prevents aatRNA binding as in the wt situation. Altogether, the data lead to the following novel resistance scenario. Upon arrival of the mutant EF-Tu·GTP·GE2270·aatRNA complex at the ribosomal A-site, the GTPase centre is triggered. The affinities of aatRNA and GE2270A for the GDP-bound EF-Tu are negligible; the former stays at the A-site for subsequent interaction with the peptidyltransferase centre and the latter two dissociate from the ribosome.

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.

Interaction of N-tosyl-L-phenylalanylchloromethane with Thermus thermophilus elongation factor Tu.

The interaction of N-tosyl-L-phenylalanylchloromethane (TosPheCH2Cl) with Thermus thermophilus elongation factor Tu (EF-Tu) was studied by affinity labelling and NMR spectroscopy. TosPheCH2Cl binds to GDP and GTP conformers of EF-Tu. The interaction of TosPheCH2Cl with EF-Tu x GDP leads to alkylation of Cys82, while interaction of TosPheCH2Cl with EF-Tu x GTP does not lead to covalent labelling. [A82]EF-Tu, in which the Cys82 is replaced by Ala, has similar properties to wild-type EF-Tu with respect to GTPase activity, binding of guanine nucleotides, interaction with elongation factor Ts (EF-Ts) and interaction with ribosomes. This structural change did not lead to changes, compared with wild-type EF-Tu in the functionality of [A82]EF-Tu, either in the GTP or in the GDP conformation. TosPheCH2Cl binds to EF-Tu x GTP with a dissociation constant of 10 microM. The interaction of TosPheCH2Cl with EF-Tu promotes the hydration of the carbonyl group of TosPheCH2Cl. TosPheCH2Cl competes with aminoacyl-tRNA for its binding site on EF-Tu x GTP. Covalent modification of Cys82 by TosPheCH2Cl does not prevent nucleotide binding and GTPase activity, but interferes with the interaction with aminoacyl-tRNA. TosPheCH2Cl probably mimics the aminoacyl residue of the aminoacyl-tRNA and binds to its binding site on EF-Tu x GTP. This rather specific interaction with EF-Tu x GTP does not allow the modification of Cys82, whereas the loose interaction of TosPheCH2Cl with EF-Tu x GDP leads to alkylation of this residue.

The G222D mutation in elongation factor Tu inhibits the codon-induced conformational changes leading to GTPase activation on the ribosome.

Elongation factor Tu (EF-Tu) from Escherichia coli carrying the mutation G222D is unable to hydrolyze GTP on the ribosome and to sustain polypeptide synthesis at near physiological Mg2+ concentration, although the interactions with guanine nucleotides and aminoacyl-tRNA are not changed significantly. GTPase and polypeptide synthesis activities are restored by increasing the Mg2+ concentration. Here we report a pre-steady-state kinetic study of the binding of the ternary complexes of wild-type and mutant EF-Tu with Phe-tRNA(Phe) and GTP to the A site of poly(U)-programed ribosomes. The kinetic parameters of initial binding to the ribosome and subsequent codon-anticodon interaction are similar for mutant and wild-type EF-Tu, independent of the Mg2+ concentration, suggesting that the initial interaction with the ribosome is not affected by the mutation. Codon recognition following initial binding is also not affected by the mutation. The main effect of the G222D mutation is the inhibition, at low Mg2+ concentration, of codon-induced structural transitions of the tRNA and, in particular, their transmission to EF-Tu that precedes GTP hydrolysis and the subsequent steps of A-site binding. Increasing the Mg2+ concentration to 10 mM restores the complete reaction sequence of A-site binding at close to wild-type rates. The inhibition of the structural transitions is probably due to the interference of the negative charge introduced by the mutation with negative charges either of the 3' terminus of the tRNA, bound in the vicinity of the mutated amino acid in domain 2 of EF-Tu, or of the ribosome. Increasing the Mg2+ concentration appears to overcome the inhibition by screening the negative charges.

Phosphorylation of Elongation Factor Tu Prevents Ternary Complex Formation

The elongation factor Tu (EF-Tu) is a member of the GTP/GDP-binding proteins and interacts with various partners during the elongation cycle of protein biosynthesis thereby mediating the correct binding of amino-acylated transfer RNA (aa-tRNA) to the acceptor site (A-site) of the ribosome. After GTP hydrolysis EF-Tu is released in its GDP-bound state. In vivo, EF-Tu is post-translationally modified by phosphorylation. Here we report that the phosphorylation of EF-Tu by a ribosome associated kinase activity is drastically enhanced by EF-Ts. The antibiotic kirromycin, known to block EF-Tu function, inhibits the modification. This effect is specific, since kirromycin-resistant mutants do become phosphorylated in the presence of the antibiotic. On the other hand, phosphorylated wild-type EF-Tu does not bind kirromycin. Most interestingly, the phosphorylation of EF-Tu abolishes its ability to bind aa-tRNA. In the GTP conformation the site of modification is located at the interface between domains 1 and 3 and is involved in a strong interdomain hydrogen bond. Introduction of a charged phosphate group at this position will change the interaction between the domains, leading to an opening of the molecule reminiscent of the GDP conformation. A model for the function of EF-Tu phosphorylation in protein biosynthesis is presented.

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.

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.

Isolation and functional analysis of histidine-tagged elongation factor Tu.

The study of the structure/function relationships of the Escherichia coli elongation factor Tu (EF-Tu) via mutagenesis has been hampered by difficulties encountered in separating the mutated factor from other proteins, in particular native EF-Tu. Here we describe a novel system for the purification of EF-Tu mutant species, based on metal-ion affinity chromatography. To facilitate rapid and efficient purification we designed a recombinant EF-Tu with an additional C-terminal sequence of one serine and six histidine residues. A cell extract containing the His-tagged EF-Tu (EF-TuHis) is applied to a Ni(2+)-nitrilotriacetic acid column. EF-TuHis can be selectively eluted with an imidazole containing buffer, yielding a preparation of more than 95% purity, free of wild-type EF-Tu. In-vitro and in-vivo functional analyses show that EF-TuHis resembles the wild-type EF-Tu, which makes this one-step isolation procedure a promising tool for the study of the interactions of mutant EF-Tu with the various components of the elongation cycle. The new isolation procedure was successfully applied for the purification of a mutant EF-TuHis with a Glu substitution for Lys237, a residue possibly involved in the binding of aminoacyl-tRNA.

Structure-function relationships of elongation factor Tu as studied by mutagenesis

We have modified elongation factor Tu (EF-Tu) from Escherichia coli via mutagenesis of its encoding tufA gene to study its function-structure relationship. The isolation of the N-terminal half molecule of EF-Tu (G domain) has facilitated the analysis of the basic EF-Tu activities, since the G domain binds the substrate GTP/GDP, catalyzes the GTP hydrolysis and is not exposed to the allosteric constraints of the intact molecule. So far, the best studied region has been the guanine nucleotide-binding pocket defined by the consensus elements typical for the GTP-binding proteins. In this area most substitutions were carried out in the G domain and were found to influence GTP hydrolysis. In particular, the mutation VG20 (in both G domain and EF-Tu) decreases this activity and enhances the GDP to GTP exchange; PT82 induces autophosphorylation of Thr82 and HG84 strongly affects the GTPase without altering the interaction with the substrate. SD173, a residue interacting with (O)6 of the guanine, abolishes the GTP and GDP binding activity. Substitution of residues Gln114 and Glu117, located in the proximity of the GTP binding pocket, influences respectively the GTPase and the stability of the G domain, whereas the double replacement VD88/LK121, located on α-helices bordering the GTP-binding pocket, moderately reduces the stability of the G domain without greatly affecting GTPase and interaction type EF-Tu, probably due to a longer pausing on the ribosome. Hyperaccuracy has also been observed with EF-TuGD222, which as a consequence of the altered interaction with the ribosome displays a recessive kirromycin-sensitivity, differently from wild-type EF-Tu. Resistance to kirromycin is associated with the substitution of Ala375 located in the C-terminal domain on the interface with the N-terminal domain. Of the two mutations obtained, AV375 and AT375, the former also influences the substrate interaction and the catalytic activity via long-range effects. The present state of art of structure-function relationship of EF-Tu is discussed and critically evaluated.

A mutation that alters the nucleotide specificity of elongation factor Tu, a GTP regulatory protein.

A single amino acid substitution (Asp to Asn) at position 138 of Escherichia coli elongation factor Tu (EF-Tu) was introduced in the tufA gene clone by oligonucleotide site-directed mutagenesis. The mutated tufA gene was then expressed in maxicells. The properties of [35S]methionine-labeled mutant and wild type EF-Tu were compared by in vitro assays. The Asn-138 mutation greatly reduced the protein's affinity for GDP; however, this mutation dramatically increased the protein's affinity for xanthosine 5'-diphosphate. The mutant protein forms a stable complex with Phe-tRNA and xanthosine 5'-triphosphate, which binds to ribosomes, whereas it does not form a complex with Phe-tRNA and GTP (10 microM). These results suggest that in EF-Tu.nucleoside diphosphate complexes, amino acid residue 138 must interact with the substituent on C-2 of the purine ring. Thus, in wild type EF-Tu, Asp-138 would hydrogen bond to the 2-amino group of GDP, and in the mutant EF-Tu, Asn-138 would form an equivalent hydrogen bond with the 2-carbonyl group of xanthosine 5'-diphosphate. Aspartic acid 138 is conserved in the homologous sequences of all GTP regulatory proteins. This mutation would allow one to specifically alter the nucleotide specificity of other GTP regulatory proteins.

An Escherichia coli mutant with an altered elongation factor Tu.

A thermosensitive mutant of Escherichia coli has been isolated that is unable to replicate the bacteriophage MS2 at 42 degrees but permits phage production at 37 degrees . Thermal inactivation studies of the supernatant enzymes show that this mutant contains a factor essential for the polymerization of phenylalanine from phenylalanyl-tRNA that at 50 degrees is more rapidly inactivated than the corresponding wild-type factor. The elongation factor Tu (EF-Tu) was isolated and purified to apparent homogeneity as the EF-Tu.GDP complex, both from mutant and wild-type cells. Addition of purified wild-type EF-Tu.GDP to reaction mixtures fully restored the activity of thermally inactivated mutant supernatants. These experiments excluded EF-Ts as the thermolabile factor involved. Similar inactivation studies, dealing with the purified factors and performed in reaction mixtures that were not supplemented with GDP, revealed that the half-life of mutant EF-Tu.GDP at 50 degrees was 1.5 min, that of the wild-type factor 6 min. Addition of GDP (10muM) to the medium reduced the inactivation rate of both wild-type and mutant factor and also the difference in inactivation kinetics. Besides the altered elongation factor Tu, the mutant skill contains a second mutation affecting the glutaminyl-tRNA synthetase.