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.     

Interaction of apicoplast-encoded elongation factor (EF) EF-Tu with nuclear-encoded EF-Ts mediates translation in the Plasmodiumfalciparum plastid

Protein translation in the plastid (apicoplast) of Plasmodium spp. is of immense interest as a target for potential anti-malarial drugs. However, the molecular data on apicoplast translation needed for optimisation and development of novel inhibitors is lacking. We report characterisation of two key translation elongation factors in Plasmodium falciparum, apicoplast-encoded elongation factor PfEF-Tu and nuclear-encoded PfEF-Ts. Recombinant PfEF-Tu hydrolysed GTP and interacted with its presumed nuclear-encoded partner PfEF-Ts. The EF-Tu inhibitor kirromycin affected PfEF-Tu activity in vitro, indicating that apicoplast EF-Tu is indeed the target of this drug. The predicted PfEF-Ts leader sequence targeted GFP to the apicoplast, confirming that PfEF-Ts functions in this organelle. Recombinant PfEF-Ts mediated nucleotide exchange on PfEF-Tu and homology modeling of the PfEF-Tu:PfEF-Ts complex revealed PfEF-Ts-induced structural alterations that would expedite GDP release from PfEF-Tu. Our results establish functional interaction between two apicoplast translation factors encoded by genes residing in different cellular compartments and highlight the significance of their sequence/structural differences from bacterial elongation factors in relation to inhibitor activity. These data provide an experimental system to study the effects of novel inhibitors targeting PfEF-Tu and PfEF-Tu.PfEF-Ts interaction. Our finding that apicoplast EF-Tu possesses chaperone-related disulphide reductase activity also provides a rationale for retention of the tufA gene on the plastid genome.     

Ef-Ts elongation factor interacts with elongation factor EF-Tu on ribosomes prior to the GTP hydrolysis stage

Methods of high-speed centrifugation and limited proteolysis were used to probe the interaction of EF-Tu with EF-Ts on the ribosome. It is shown that EF-Ts dissociates from EF-Tu only after EF-Tu-mediated GTP hydrolysis, i.e. EF-Ts within the EF-Tu.ribosome complexes in the pre-GTP-hydrolysis state co-sediments with the ribosomes and its rate of proteolysis is distinct from that of free EF-Ts. Moreover, as seen from the difference in sensitivity to trypsin of ribosomal proteins L19 and L27 EF-Ts affects the interaction of EF-Tu with the ribosome.     

Methylation in vivo of elongation factor EF-Tu at lysine-56 decreases the rate of tRNA-dependent GTP hydrolysis

In this paper we show, that the in vivo methylation of the elongation factor Tu from Escherichia coli is correlated with the growth phase of the bacterium. Methylation occurs at one position only, i.e. Lys-56, and initially results in monomethylation during logarithmic growth. Upon entering the stationary phase of E. coli, monomethyllysine is gradually converted into dimethyllysine. We have undertaken an extensive comparison between the properties of the highly methylated EF-Tu and unmodified EF-Tu. No gross conformational differences, as measured by the rate of mild tryptic cleavage, were observed. The dissociation rates of the nucleotides GDP and GTP appear likewise to be unaffected by the methylation, just as is the stimulatory effect of the elongation factor Ts upon these rates. Whereas tRNA binding at the classical binding site of EF-Tu (site I) also appears not to be affected by the methylation of the protein, tRNA binding at site II is. Although the apparent affinity of tRNA for site II remains unaltered upon methylation of EF-Tu, the conformational effects of tRNA binding at this site become different. Both the GTPase activity of the protein and the reactivity of Cys-81 are significantly less stimulated by the tRNA when EF-Tu is methylated. A possible physiological implication of this phenomenon is discussed.     

Outwitting EF-Tu and the ribosome: translation with d-amino acids

Key components of the translational apparatus, i.e. ribosomes, elongation factor EF-Tu and most aminoacyl-tRNA synthetases, are stereoselective and prevent incorporation of d-amino acids (d-aa) into polypeptides. The rare appearance of d-aa in natural polypeptides arises from post-translational modifications or non-ribosomal synthesis. We introduce an in vitro translation system that enables single incorporation of 17 out of 18 tested d-aa into a polypeptide; incorporation of two or three successive d-aa was also observed in several cases. The system consists of wild-type components and d-aa are introduced via artificially charged, unmodified tRNA^Gly that was selected according to the rules of ‘thermodynamic compensation’. The results reveal an unexpected plasticity of the ribosomal peptidyltransferase center and thus shed new light on the mechanism of chiral discrimination during translation. Furthermore, ribosomal incorporation of d-aa into polypeptides may greatly expand the armamentarium of in vitro translation towards the identification of peptides and proteins with new properties and functions.     

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.     

A simplified procedure for the isolation of bacterial polypeptide elongation factor EF-Tu

Polypeptide elongation factor EF-Tu can be isolated from bacterial cell extracts in two fractionation steps. The first is ion-exchange chromatography on DEAE-Sepharose, CL-6B, and the second is gel filtration on AcA 44. The method is illustrated with extracts from Escherichia coli, Bacillus stearothermophilus, and the thermophilic bacterium PS3. The extracts were obtained from lysozyme-treated cells and were processed without high-speed centrifugation or ammonium sulfate fractionation. The procedure is simple and rapid, gives higher yields than previous methods, and is easily scaled to any size preparation. The procedure also produces fractions enriched in the other polypeptide elongation factors EF-Ts and EF-G.     

Trade-offs between tRNA abundance and mRNA secondary structure support smoothing of translation elongation rate

Translation of protein from mRNA is a complex multi-step process that occurs at a non-uniform rate. Variability in ribosome speed along an mRNA enables refinement of the proteome and plays a critical role in protein biogenesis. Detailed single protein studies have found both tRNA abundance and mRNA secondary structure as key modulators of translation elongation rate, but recent genome-wide ribosome profiling experiments have not observed significant influence of either on translation efficiency. Here we provide evidence that this results from an inherent trade-off between these factors. We find codons pairing to high-abundance tRNAs are preferentially used in regions of high secondary structure content, while codons read by significantly less abundant tRNAs are located in lowly structured regions. By considering long stretches of high and low mRNA secondary structure in Saccharomyces cerevisiae and Escherichia coli and comparing them to randomized-gene models and experimental expression data, we were able to distinguish clear selective pressures and increased protein expression for specific codon choices. The trade-off between secondary structure and tRNA-concentration based codon choice allows for compensation of their independent effects on translation, helping to smooth overall translational speed and reducing the chance of potentially detrimental points of excessively slow or fast ribosome movement.     

Refined structure of elongation factor EF-Tu from Escherichia coli.

The crystal structure of trypsin-modified elongation factor Tu from Escherichia coli, in complex with the cofactor guanosine diphosphate has been refined to a crystallographic R-factor of 19.3%, at 2.6 A resolution. In the model described, the root-mean-square deviation from ideality is 0.019 A for bond distances and 3.9 degrees for angles. The protein consists of three domains: an alpha/beta domain (residues 1 to 200), containing the binding site of the GDP cofactor, and consisting of a six-stranded beta-pleated sheet, six alpha-helices, and two all-beta domains (residues 209 to 299 and 300 to 393), belonging to the tertiary structural class of antiparallel beta-barrels. The GDP-binding domain has a folding that is found in other GDP-binding proteins. Elongation factor Tu interacts with proteins, nucleic acids and nucleotides, making this molecule well suited as a model system for the study of these interactions.     

Evidence for activities of rabbit reticulocyte elongation factor 1 analogous to bacterial factors EF-Ts and EF-Tu

Preparations have been obtained from rabbit reticulocyte elongation factor 1 (EF-1) that exhibit activities analogous to the heat stable and heat labile factors, EF-Ts and EF-Tu, of $\text{Escherichia coli}$. The heat stable fraction, prepared by heating EF-1 in the presence of GTP, has virtually no activity in poly (U)-directed polyphenylalanine synthesis. The fraction exhibiting activity similar to bacterial EF-Tu is obtained by the interaction of EF-1 with GTP and phenylalanyl-tRNA followed by passage of the solution through a nitrocellulose filter. The filtrate, which alone has low activity in polyphenylalanine synthesis, when combined with the heat stable fraction gives high activity suggesting that the heat stable preparation catalyzes recycling of the filtrate component.     

Functional implications related to the gene structure of the elongation factor EF-Tu from Halobacterium marismortui.

The primary structure of the gene for the elongation factor EF-Tu from the halophilic archaebacterium Halobacterium marismortui (hEF-Tu) is described. It is the first gene of a halophilic elongation factor EF-Tu to be sequenced. When the sequence of hEF-Tu is compared to that of homologous proteins from other organisms, the highest identity (61%) is found with EF-Tu from Methanococcus vannielii, a non-halophilic archaebacterium. In the search for halophilic characteristics therefore the most appropriate comparison is with the M. vannielii sequence. The excess of acidic amino acid residues in the hEF-Tu sequence (already observed in the composition of other halophilic proteins) results to a large extent from changes of Lys, Asn or Gln to Asp or Glu. A structural analysis algorithm applied to the halophilic sequence places these acidic residues on the surface of the protein. The corresponding residues in the crystal structure of the first domain of EF-Tu from E. coli (the only EF-Tu structure available) are grouped in patches on the protein surface, in each of which several residues that may be far apart in the sequence come quite close to each other in the tertiary structure.     

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.     

Isolation of chimaeric forms of elongation factor EF-Tu by affinity chromatography

Six different recombinant chimaeric forms of a three-domain protein, proteosynthetic elongation factor Tu (EF-Tu), composed of domains of EF-Tu of mesophilic (Escherichia coli) and thermophilic (Bacillus stearothermophilus) origin as well as free N-terminal domains of EF-Tu, and the whole recombinant EF-Tus of both organisms were prepared and isolated by the GST (glutathione S-transferase) fusion technology. Several modifications in the standard isolation and purification procedures are described that proved necessary to obtain the proteins in a purified and undegraded form.     

Cloning and characterization of the chloroplast elongation factor EF-Tu cDNA of Oryza sativa L

From the rice leaf cDNA library, we have cloned a cDNA encoding rice chloroplast translational elongation factor EF-Tu (tufA). The rice tufA cDNA clone contains 1678 nucleotides and codes for a 467 amino acid protein including a putative chloroplast transit peptide of 59 amino acid residues. The predicted molecular mass of the mature protein is approximately 45 kDa. This cDNA clone contains the 61 nucleotides of the 5' untranslated region (UTR) and the 213 nucleotides of 3' UTR. Amino acid sequence identity of the rice tufA with the mature chloroplast EF-Tu proteins of tobacco, pea, arabidopsis, and soybean ranges from 83% to 86%. The deduced polypeptide of the rice tufA cDNA contains GTP binding domains in its N-terminal region and chloroplast EF-Tu signature regions in the C-terminal region. The rice tufA appears to exist as a single copy gene, although its homologues of maize and oat exist as multiple copy genes. The rice tufA gene is located in chromosome 1 and is more highly expressed in the leaf than in root tissue.     

Effects of mutagenesis of residue 221 on the properties of bacterial and mitochondrial elongation factor EF-Tu

During protein biosynthesis, elongation factor Tu (EF-Tu) delivers aminoacyl-tRNA (aa-tRNA) to the A-site of ribosomes. This factor is highly conserved throughout evolution. However, several key residues differ between bacterial and mammalian mitochondrial EF-Tu (EF-Tu(mt)). One such residue is Ser221 (Escherichia coli numbering). This residue is conserved as a Ser or Thr in the bacterial factors but is present as Pro269 in EF-Tu(mt). Pro269 reorients the loop containing this residue and shifts the adjoining beta-strand in EF-Tu(mt) compared to that of E. coli EF-Tu potentially altering the binding pocket for the acceptor stem of the aa-tRNA. Pro269 was mutated to a serine residue (P269S) in EF-Tu(mt). For comparison, the complementary mutation was created at Ser221 in E. coli EF-Tu (S221P). The E. coli EF-Tu S221P variant is poorly expressed in E. coli and the majority of the molecules fail to fold into an active conformation. In contrast, EF-Tu(mt) P269S is expressed to a high level in E. coli. When corrected for the percentage of active molecules, both variants function as effectively as their respective wild-type factors in ternary complex formation using E. coli Phe-tRNA(Phe) and Cys-tRNA(Cys). They are also active in A-site binding and in vitro translation assays with E. coli Phe-tRNA(Phe). In addition, both variants are as active as their respective wild-type factors in ternary complex formation, A-site binding and in vitro translation assays using mitochondrial Phe-tRNA(Phe).     

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.     

In vivo and in vitro methylation of the elongation factor EF-Tu from Euglena gracilis chloroplast

Based on amino acid sequence similarities between the methylated elongation factor EF-Tu from Escherichia coli and the EF-Tu from Euglena gracilis chloroplast, we predicted that the latter could also be methylated in the presence of an appropriate methyltransferase. We found that, as reported for the eubacterial homologous protein, the organellar factor could be methylated in vivo and in vitro to yield monomethyllysine.     

Immunological heterogeneity of archaebacterial protein synthesis elongation factors Tu (EF-Tu)

Abstract Polyclonal antibodies were raised against the EF-Tu of the archaebacterium Sulfolobus solfataricus and cross-reactivities of EF-Tus of other, phylogenetically disparate archaebacteria were determined using Western blotting and ELISA. The results demonstrate a high degree of heterogeneity of archaebacterial Tu factors with recognition by S. solfataricus EF-Tu antibodies ranging from 48% to 1.5% that observed with the homologous antigen. The immunochemical relatedness between the heterologous and the cognate ( Sulfolobus ) antigens correlates satisfactorily with similarities in 16 S rRNA sequences, there being no recognition of eubacterial and eukaryotic factors by the S. solfataricus EF-Tu antibodies.