Mitochondrial polypeptide elongation factor EF-Tu of Saccharomyces cerevisiae. Functional and structural homologies to Escherichia coli EF-Tu

The polypeptide elongation factor EF-Tu was isolated from a mitochondrial 100 000 x g supernatant of the yeast Saccharomyces cerevisiae and purified over 880-fold by DEAE-Sephadex chromatography and gel filtration. The factor efficiently replaces bacterial EF-Tu in a phenylalanine polymerizing cell-free system of Escherichia coli, it binds GDP and it protects phenylalanyl-tRNA against hydrolysis of the ester bond in the presence of 10 mM GTP. The polymerizing activity of the mitochondrial factor is inhibited to 90% by 50 microM N-ethylmaleimide and to 50% by 2.5 microM kirromycin. The purified factor contains two major polypeptides of apparent molecular weights 48 000 and 34 000. Antibodies raised against the 48 000-Mr protein react with EF-TuE. coli, as revealed by immune blotting and by the inhibition of phenylalanine polymerization. No reaction was observed between anti-(34 000-Mr) and 48 000-Mr protein or EF-TuE. coli. The 48 000-Mr protein has the same isoelectric point (pI = 6.2) and a content of cysteine and basic amino acids similar to the bacterial EF-Tu. It is concluded that the 48 000-Mr protein is the analogue to EF-TuE. coli, and that yeast mitochondrial EF-Tu is functionally and structurally more related to bacterial EF-Tu than cytosolic EF-1 of the same cell.     

Elongation factor EF-Ts interacts with the aminoacyl-tRNA.EF-Tu.GTP complex]

The fluorescence polarization technique has been used to study the interaction of the EF-Ts dansyl derivative with EF-Tu after nucleotide exchange and binding of the aminoacyl-tRNA to EF-Tu.GTP. It is shown that the ternary complex formation results in the increase of EF-Ts affinity to EF-Tu and EF-Ts remains bound to EF-Tu up to the GTP hydrolysis stage on the ribosome.     

耐热蛋白质延长因子（EF—Tu）的分子和功能的性质

Protein synthesis elongation factor Tu (EF-Tu) was purified from an extreme thermophilic hydrogen-oxidizing bacterium Calderobacterium hydrogenophilum. The relative molecular mass of EF-Tu. GDP was 51,000. The factor was heat stable and lost only 50% of its activity after heating at 80 degrees C for 5 min. Native and reduced EF-Tu or EF-Tu. GDP contained one SH-reactive group. The elongation factors from C. hydrogenophilum and E. coli were shown to be immunologically identical. From the Southern hybridization analysis seems to suggest that chromosome DNA of C. hydrogenophilum has two tuf genes.     

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.     

Efficient separation of Thermus aquaticus EF-Tu functional complexes

A new method for fast separation of the main functional complexes of the elongation factor Tu from Thermus aquaticus has been developed. Binary complexes EF-Tu * GDP and EF-Tu * GDPNP as well as the ternary complex EF-Tu * GDPNP * Leu approximately tRNA were separated from each other by means of HPLC on a hydrophobic sorbent TSK-Gel Phenyl 5PW in a reverse gradient of ammonium sulfate. This technique is suitable for monitoring EF-Tu activity, characterisation of the ratio between different EF-Tu forms in cell extracts, and isolation of individual EF-Tu complexes for structural and functional investigations. In order to illustrate the potentials of the method, we used HPLC on a TSK-Gel Phenyl 5PW matrix to determine the ratio between affinities of GDP and GDPNP for EF-Tu. We found that K(a)(GDP) is about 27 times higher than K(a)(GDPNP) at 37 degrees C, the value being close to the one reported for Thermus thermophilus EF-Tu.     

Mechanism of EF-Ts-catalyzed guanine nucleotide exchange in EF-Tu: contribution of interactions mediated by helix B of EF-Tu

Elongation factor Tu (EF-Tu) belongs to the family of GTP-binding proteins and requires elongation factor Ts (EF-Ts) for nucleotide exchange. Crystal structures suggested that one of the salient features in the EF-Tu x EF-Ts complex is a conformation change in the switch II region of EF-Tu that is initiated by intrusion of Phe81 of EF-Ts between His84 and His118 of EF-Tu and may result in a destabilization of Mg2+ coordination and guanine nucleotide release. In the present paper, the contribution of His84 to nucleotide release was studied by pre-steady-state kinetic analysis of nucleotide exchange in mutant EF-Tu in which His84 was replaced by Ala. Both intrinsic and EF-Ts-catalyzed nucleotide release was affected by the mutation, resulting in a 10-fold faster spontaneous GDP release and a 4-fold faster EF-Ts-catalyzed release of GTP and GDP. Removal of Mg2+ from the EF-Tu x EF-Ts complex increased the rate constant of GDP release 2-fold, suggesting a small contribution to nucleotide exchange. Together with published data on the effects of mutations interfering with other putative interactions between EF-Tu and EF-Ts, the results suggest that each of the contacts in the EF-Tu x EF-Ts complex alone contributes moderately to nucleotide destabilization, but together they act synergistically to bring about the overall 60,000-fold acceleration of nucleotide exchange in EF-Tu by EF-Ts.     

Isolation,crystallisation, and preliminary X-ray analysis of the bovine mitochondrial EF-Tu:GDP and EF-Tu:EF-Ts complexes

Previous studies have shown that when bovine mitochondrial elongation factor Ts (EF-Ts) is expressed in Escherichia coli, it forms a tightly associated complex with E. coli elongation factor Tu (EF-Tu). In contrast to earlier experiments, purification of free mitochondrial EF-Ts was accomplished under nondenaturing conditions since only about 60% of the expressed EF-Ts copurified with E. coli EF-Tu. The bovine mitochondrial EF-Tu:GDP complex, the homologous mitochondrial EF-Tu:EF-Ts complex, and the heterologous E. coli/mitochondrial EF-Tu:EF-Ts complex were isolated and crystallised. The crystals of the EF-Tu:GDP complex diffract to 1.94 A and belong to space group P2(1) with cell parameters a=59.09 A, b=119.78 A, c=128.89 A and beta=96.978 degrees. The crystals of the homologous mitochondrial EF-Tu:EF-Ts complex diffract to 4 A and belong to space group C2 with cell parameters a=157.7 A, b=151.9 A, c=156.9 A, and beta=108.96 degrees.     

Mutant EF-Tu increases missense error in vitro

Summary We have studied the consequences of mutational alteration in the structure of EF-Tu on the missense errors and proofreading activity of bacterial ribosomes in vitro. Our data show that the EF-Tu Bo mutant form of EF-Tu (van der Meide et al. 1983a) is inactive in polypeptide synthesis on the ribosome, even though it binds aminoacyl-tRNA. A second mutant form, EF-Tu Ar (van der Meide et al. 1983a), is active in polypeptide synthesis but supports a much higher messense incorporation with either leucine isoacceptor 2 or leucine isoacceptor 4 in the in vitro system. Further analysis of the kinetic basis of this enhanced missense frequency revealed that the mutation responsible for the alteration in EF-Tu Ar increases the errors at both the proofreading step and the initial selection. In this respect the effect of this particular mutation is similar to the mode of action of the antibiotic kanamycin (Jelenc and Kurland 1984).     

Polymerization of the bacterial elongation factor for protein synthesis, EF-Tu.

The bacterial elongation factor for protein synthesis, EF-Tu, polymerizes into fibrils at pH 6.0. These fibrils are 0.7 microM in diameter, at least 200 microns in length, and are positively birefringent. Electron microscopic observations of negatively stained images demonstrates that the EF-Tu fibrils consist of bundles of individual filaments, approximately 5nm in diameter, aligned parallel to the long axis of the fibril. Polymerized EF-Tu exchanges nucleotide rapidly and interacts with the other elongation factor, EF-Ts. The antibiotic kirromycin induces the polymerization of EF-Tu into fibrils and even larger structures under nonpolymerizing conditions.     

Interaction between initiator Met-tRNAfMet and elongation factor EF-Tu from E. coli

It has recently been shown that the non-formylated initiator Met-tRNAfMet from E. coli can form a stable ternary complex with the elongation factor EF-Tu and GTP. Using the protection of EF-Tu:GTP against spontaneous hydrolysis of the aminoacylester bond of Met-tRNAfMet, we confirm these results, and show that the protection is specific for the non-formylated form of the initiator tRNA. The ternary complex Met-tRNAfMet:EF-Tu:GTP can be isolated by column chromatography in a way similar to that demonstrated previously with EF-Tu complexed to the elongator Met-tRNAmMet. 32P-labeled Met-tRNAfMet within the ternary complex was analyzed by the footprinting technique. The pattern of initiator tRNA protection by EF-Tu against ribonuclease digestion is not significantly different from the one found previously for elongator tRNAs. These results lead us to suggest that the initiator tRNAfMet, under growth conditions which do not permit formylation, may to some extent function as an elongator tRNA.     

EF-Tu dynamics during pre-translocation complex formation: EF-Tu·GDP exits the ribosome via two different pathways

The G-protein EF-Tu, which undergoes a major conformational change when EF-Tu·GTP is converted to EF-Tu·GDP, forms part of an aminoacyl(aa)-tRNA·EF-Tu·GTP ternary complex (TC) that accelerates the binding of aa-tRNA to the ribosome during peptide elongation. Such binding, placing a portion of EF-Tu in contact with the GTPase Associated Center (GAC), is followed by GTP hydrolysis and P_i release, and results in formation of a pretranslocation (PRE) complex. Although tRNA movement through the ribosome during PRE complex formation has been extensively studied, comparatively little is known about the dynamics of EF-Tu interaction with either the ribosome or aa-tRNA. Here we examine these dynamics, utilizing ensemble and single molecule assays employing fluorescent labeled derivatives of EF-Tu, tRNA, and the ribosome to measure changes in either FRET efficiency or fluorescence intensity during PRE complex formation. Our results indicate that ribosome-bound EF-Tu separates from the GAC prior to its full separation from aa-tRNA, and suggest that EF-Tu·GDP dissociates from the ribosome by two different pathways. These pathways correspond to either reversible EF-Tu·GDP dissociation from the ribosome prior to the major conformational change in EF-Tu that follows GTP hydrolysis, or irreversible dissociation after or concomitant with this conformational change.     

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.     

Missense substitutions lethal to essential functions of EF-Tu

We have used a simple selection and screening method to isolate function defective mutants of EF-Tu. From 28 mutants tested, 12 different missense substitutions, individually lethal to some essential function of EF-Tu, were identified by sequencing. In addition we found a new non-lethal missense mutation. The frequency of isolation of unique mutations suggests that this method can be used to easily isolate many more. The lethal mutations occur in all three structural domains of EF-Tu, but most are in domain II. We aim to use these mutants to define functional domains on EF-Tu.     

A unique tRNA recognition mechanism of Caenorhabditis elegans mitochondrial EF-Tu2

Nematode mitochondria expresses two types of extremely truncated tRNAs that are specifically recognized by two distinct elongation factor Tu (EF-Tu) species named EF-Tu1 and EF-Tu2. This is unlike the canonical EF-Tu molecule that participates in the standard protein biosynthesis systems, which basically recognizes all elongator tRNAs. EF-Tu2 specifically recognizes Ser-tRNA^Ser that lacks a D arm but has a short T arm. Our previous study led us to speculate the lack of the D arm may be essential for the tRNA recognition of EF-Tu2. However, here, we showed that the EF-Tu2 can bind to D arm-bearing Ser-tRNAs, in which the D–T arm interaction was weakened by the mutations. The ethylnitrosourea-modification interference assay showed that EF-Tu2 is unique, in that it interacts with the phosphate groups on the T stem on the side that is opposite to where canonical EF-Tu binds. The hydrolysis protection assay using several EF-Tu2 mutants then strongly suggests that seven C-terminal amino acid residues of EF-Tu2 are essential for its aminoacyl-tRNA-binding activity. Our results indicate that the formation of the nematode mitochondrial (mt) EF-Tu2/GTP/aminoacyl-tRNA ternary complex is probably supported by a unique interaction between the C-terminal extension of EF-Tu2 and the tRNA.     

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.     

The influence of tRNA located at the P-site on the turnover of EF-Tu.GTP on ribosomes.

The turnover of EF-Tu.GTP on poly-U programmed ribosomes was measured both in the presence and in the absence of N-acetylated Phe-tRNA(Phe) at the P-site. The reaction was uncoupled from protein synthesis by omitting Phe-tRNA(Phe) at the A-site. In this reaction, the ribosome can be considered as an enzyme catalysing the transition of EF-Tu.GTP to EF-Tu.GTP. A constant EF-Tu.GTP concentration is maintained by regenerating GDP to GTP at the expense of phosphoenolpyruvate by pyruvate kinase. The rate constants are determined using a procedure which corrects for the reduction in specific activity of GTP due to regeneration of the nucleotide. Ribosomes with an occupied P-site are more efficient in stimulating the GTPase of EF-Tu.GTP than ribosomes with an empty P-site. The data suggest that this is mainly caused by an increased affinity of EF-Tu.GTP for ribosomes with a filled P-site rather than by an enhanced reactivity of the GTPase centre.     

Effect of GDP on the interactions between chloroplast EF-Ts and chloroplast and E. coli EF-Tu

The effects of varying concentrations of GDP on the stability of homologous and heterologous EF-Tu:EF-Ts complexes formed with the elongation factors from the chloroplast of Euglena gracilis and from E. coli have been investigated. The complexes formed with chloroplast EF-Ts were significantly more stable to GDP-induced dissociation than those formed with E. coli EF-Ts. The complex between chloroplast EF-Tu and chloroplast EF-Ts required nearly 1,000-fold higher concentrations of GDP for dissociation than the complex between chloroplast EF-Tu and E. coli EF-Ts. The E. coli EF-Tu:chloroplast EF-Ts complex required nearly 100-fold higher levels of GDP for dissociation than the E. coli EF-Tu:E. coli EF-Ts complex.     

Chaperone activity of recombinant maize chloroplast protein synthesis elongation factor, EF-Tu.

The protein synthesis elongation factor, EF-Tu, is a protein that carries aminoacyl-tRNA to the A-site of the ribosome during the elongation phase of protein synthesis. In maize (Zea mays L) this protein has been implicated in heat tolerance, and it has been hypothesized that EF-Tu confers heat tolerance by acting as a molecular chaperone and protecting heat-labile proteins from thermal aggregation and inactivation. In this study we investigated the effect of the recombinant precursor of maize EF-Tu (pre-EF-Tu) on thermal aggregation and inactivation of the heat-labile proteins, citrate synthase and malate dehydrogenase. The recombinant pre-EF-Tu was purified from Escherichia coli expressing this protein, and mass spectrometry confirmed that the isolated protein was indeed maize EF-Tu. The purified protein was capable of binding GDP (indicative of protein activity) and was stable at 45 degrees C, the highest temperature used in this study to test this protein for possible chaperone activity. Importantly, the recombinant maize pre-EF-Tu displayed chaperone activity. It protected citrate synthase and malate dehydrogenase from thermal aggregation and inactivation. To our knowledge, this is the first observation of chaperone activity by a plant/eukaryotic pre-EF-Tu protein. The results of this study support the hypothesis that maize EF-Tu plays a role in heat tolerance by acting as a molecular chaperone and protecting chloroplast proteins from thermal aggregation and inactivation.     

Cold-stress-altered phosphorylation of EF-Tu in the cyanobacterium Anabaena sp. strain PCC 7120

Growth of prokaryotes at reduced temperature results in the formation of a cold-adapted ribosome through association with de novo synthesized polypeptides. In vitro and in vivo phosphorylation studies combined with affinity purification and mass spectrometry identified that the phosphorylation status of translation elongation factor EF-Tu was altered in response to cold stress in the photosynthetic, Gram-negative cyanobacterium Anabaena sp. strain PCC 7120. In response to a temperature downshift from 30 to 20 degrees C, EF-Tu was rapidly and transiently hyperphosphorylated during the acclimation phase followed by a reduction in phosphorylation below background levels in response to prolonged exposure. EF-Tu was identified as a phosphothreonine protein. Unexpectedly, ribosomal protein S2 was also observed to be a phosphoprotein continuously phosphorylated during cold stress. The phosphorylation status of EF-Tu has previously been associated with translational regulation in other systems, with a reduction in translation elongation occurring in response to phosphorylation. These results provide evidence for a novel mechanism by which translation is initially downregulated in response to cold stress in Anabaena.