The isolation and mapping of EF-Tu mutations in Salmonella typhimurium

Summary The first isolation of EF-Tu mutations in Salmonella typhimurium is reported. The mutations were isolated by selecting for resistance to the antibiotic mocimycin (= kirromycin). The mocimycin resistant phenotype is the result of mutations in each of two genes, tufA and tufB. Strains mutant in only one of the two tuf genes are sensitive to mocimycin. The spontaneous mutation rate of each of the two tuf genes to a mocimycin resistant phenotype differs by an order of magnitude. tufA maps at minute 71–72, closely linked to rpsL. tufB maps at minute 88–89, closely linked to rpoB. These map positions correspond to the locations of tufA and tufB in E. coli.Abbreviations EF-Tu protein elongation factor Tu - MOC mocimycin     

Either of the chromosomal tuf genes of E. coli K-12 can be deleted without loss of cell viability

A method of λ-mediated gene replacement was used to disrupt tufA or tufB on the chromosome of the E. coli K-12 strain MG1655. Both tuf genes, which are almost identical but map in different chromosomal contexts, encode the essential peptide chain elongation factor EF-Tu, one of the most abundant cytoplasmic proteins. Southern analysis confirmed replacement of the chromosomal tufA or tufB gene by a chloramphenicol resistance marker, demonstrating that both tuf genes are individually dispensable for growth. Under conditions of rapid growth, deletion of tufB had no significant effect on growth rate, but deletion of tufA resulted in a 35% increase in generation time. In minimal medium we observed no negative effects of tufA deletion on growth rate. Strains with a single tuf gene are useful for the expression of mutant forms of EF-Tu as the sole species in cells; this was demonstrated by introducing the hybrid tufAhis gene, encoding EF-TuA extended with a C-terminal (His)₆ tag, into the chromosome of a strain lacking tufB. Received: 15 July 1998 / Accepted: 13 October 1998     

Either of the chromosomal tuf genes of E. coli K-12 can be deleted without loss of cell viability

A method of λ-mediated gene replacement was used to disrupt tufA or tufB on the chromosome of the E. coli K-12 strain MG1655. Both tuf genes, which are almost identical but map in different chromosomal contexts, encode the essential peptide chain elongation factor EF-Tu, one of the most abundant cytoplasmic proteins. Southern analysis confirmed replacement of the chromosomal tufA or tufB gene by a chloramphenicol resistance marker, demonstrating that both tuf genes are individually dispensable for growth. Under conditions of rapid growth, deletion of tufB had no significant effect on growth rate, but deletion of tufA resulted in a 35% increase in generation time. In minimal medium we observed no negative effects of tufA deletion on growth rate. Strains with a single tuf gene are useful for the expression of mutant forms of EF-Tu as the sole species in cells; this was demonstrated by introducing the hybrid tufAhis gene, encoding EF-TuA extended with a C-terminal (His)₆ tag, into the chromosome of a strain lacking tufB.     

Characterization of the interaction between the nucleotide exchange factor EF-Ts from nematode mitochondria and elongation factor Tu

Caenorhabditis elegans mitochondria have two elongation factor (EF)-Tu species, denoted EF-Tu1 and EF-Tu2. Recombinant nematode EF-Ts purified from Escherichia coli bound both of these molecules and also stimulated the translational activity of EF-Tu, indicating that the nematode EF-Ts homolog is a functional EF-Ts protein of mitochondria. Complexes formed by the interaction of nematode EF-Ts with EF-Tu1 and EF-Tu2 could be detected by native gel electrophoresis and purified by gel filtration. Although the nematode mitochondrial (mt) EF-Tu molecules are extremely unstable and easily form aggregates, native gel electrophoresis and gel filtration analysis revealed that EF-Tu·EF-Ts complexes are significantly more soluble. This indicates that nematode EF-Ts can be used to stabilize homologous EF-Tu molecules for experimental purposes. The EF-Ts bound to two eubacterial EF-Tu species (E.coli and Thermus thermophilus). Although the EF-Ts did not bind to bovine mt EF-Tu, it could bind to a chimeric nematode–bovine EF-Tu molecule containing domains 1 and 2 from bovine mt EF-Tu. Thus, the nematode EF-Ts appears to have a broad specificity for EF-Tu molecules from different species.     

Rates of growth, ribosome synthesis and elongation factor synthesis in a tufA defective strain of E. coli

Summary A tufA defective strain of E. coli was isolated which by a single deletion event acquired a tufA-lacZ fusion gene and lost the normal functional tufA gene (see accompanying paper). A correlation between the growth rate and the rate of ribosome synthesis showed that the average rate of protein synthesis was decreased to about 50% in the tufA defective strain whereas the number of EF-Tu molecules per ribosome was about 80% compared to a normal strain. The results indicate that tufB gene expression was preferentially stimulated in the tufA defective strain but the increased EF-TuB synthesis was not sufficient to make up for the loss of normal EF-TuA synthesis. Introduction of a plasmid that carries a complete tufA gene and the preceeding fusA gene but not the str-promotor into the tufA defective strain did not alleviate the slow growth or low rate of EF-Tu synthesis showing that the high rate of EF-TuA synthesis compared to the other proteins in the str operon is not augmented by a strong second promotor for the tufA gene. The tufA-lacZ fusion which takes the place of the normal tufA gene was expressed at a high rate and the -galactosidase activity increased with the growth rate as expected.     

A mutant of Escherichia coli blocked in peptide elongation: altered elongation factor Ts

Among our transfer RNA-dependent growth mutants, one, HAK88, was found that carries an altered elongation factor Ts. The activity of mutant EFTs to bind GDP to EFTu, or to form the ternary complex (aminoacyl-tRNA-EFTu-GTP) is thermolabile. The effect of magnesium on the formation of EFTu-GDP from the EFTu-EFTs complex of HAK8 shows that a four to fivefold increase of the duplex formation occurs when the magnesium concentration is increased from 10^?6m to 10^?2m at 0 °C and at 41 °C. However, at higher temperatures, formation of the binary EFTu-GDP from the EFTu-EFTs complex of HAK88 is depressed, even at 10^?3m to 10^?2m-magnesium. The binding of GDP to the wild-type or mutant EFTu-EFTs complex at 0 °C and 42 °C indicates that the formation of EFTu-GDP is inhibited at 42 °C only when mutant complex is used for the assay. Binding of GTP to complete bacteriophage Qβ replicase (which is known to contain EFTs) formed in phage-infected HAK88 is also inhibited at 42 °C.     

Bacterial elongation factor Ts: isolation and reactivity with elongation factor Tu.

An improved method for the purification of bacterial polypeptide elongation factor Ts (EF-Ts) from one mesophile (Escherichia coli) and two thermophiles (Bacillus stearothermophilus and PS3) is described. The improvements are both in the facility of isolation and in increased yields. The purified factors were used for cross-reactivity studies with elongation factor Tu (EF-Tu) obtained from the same bacterial strains. In all combinations studied, the efficiency of EF-Ts in catalyzing the exchange of EF-Tu-bound GDP was proportional to the strength of the protein-protein complex. Whereas the factors from the two thermophiles were interchangeable, the mesophilic EF-Ts formed a very weak complex with thermophilic EF-Tu; however, thermophilic EF-Ts formed very strong complexes with mesophilic EF-Tu. Thus, e.g., EF-Tu from E. coli formed a complex with EF-Ts from B. stearothermophilus which was 10 times more stable than the corresponding homologous complex.     

Effect of Thermus thermophilus elongation factor Ts on the conformation of elongation factor Tu

Affinity labeling in situ of the Thermus thermophilus elongation factor Tu (EF-Tu) nucleotide binding site was achieved with periodate-oxidized GDP (GDPoxi) or GTP (GTPoxi) in the absence and presence of elongation factor Ts (EF-Ts). Lys52 and Lys137, both reacting with GDPoxi and GTPoxi, are located in the nucleotide binding region. In the absence of EF-Ts Lys137 and to a lesser extent Lys52 were accessible to the reaction with GTPoxi. GDPoxi reacted much more efficiently with Lys52 than with Lys137 under these conditions [Peter, M. E., Wittman-Liebold, B. & Sprinzl, M. (1988) Biochemistry 27, 9132-9138]. In the presence of EF-Ts, GDPoxi reacted more efficiently with Lys137 than with Lys52, indicating that the interaction of EF-Ts with EF-Tu.GDPoxi induces a conformation resembling that of the EF-Tu.GDPoxi complex in the absence of EF-Ts. Binding of EF-Ts to EF-Tu.GDP enhances the accessibility of the Arg59-Gly60 peptide bond of EF-Tu to trypsin cleavage. Hydrolysis of this peptide bond does not interfere with the ability of EF-Ts to bind to EF-Tu. EF-Ts is protected against trypsin cleavage by interaction with EF-Tu.GDP. High concentrations of EF-Ts did not interfere significantly with aminoacyl-tRNA.EF-Tu.GTP complex formation.     

Gene expression of chloroplast translation elongation factor Tu during maize chloroplast biogenesis

We have examined the expression of a maize nucleartuf gene(tufA) coding for the chloroplast translation elongation factor EF-Tu. Southern analysis revealed that the maize chloroplast EF-Tu was encoded by at least two distinct genes in the nuclear genome. In order to know the effect of light on the expression of thetufA gene during maize chloroplast biogenesis, we have analyzed the steady-state level of thetufA mRNAs by Northern analysis. The steady-state level of thetufA mRNAs was similar in both continuous light- and dark-grown seedlings. The level of thetufA mRNAs also maintained at relatively same level during light-induced greening of etiolated seedlings and all examined developmental stages. These results indicate that the gene expression of the maize chloroplast EF-Tu is rarely light-regulated at it’s mRNA level during chloroplast biogenesis.     

Catalytic effects of elongation factor Ts on polypeptide synthesis

The kinetic parameters which characterize the interaction between elongation factor Tu (EF-Tu) and elongation factor Ts (EF-Ts) have been determined in a poly(uridylic acid)-primed translation system. The EF-Ts catalyzed release of GDP from EF-Tu was measured independently in a nucleotide exchange assay. We conclude that the rate-limiting step for the EF-Tu cycle in protein synthesis in the absence of EF-Ts is the release of GDP. By adding EF-Ts the time of this step is reduced from 90 s to 30 ms. Half maximal rate is obtained at an EF-Ts concentration of 2.5 x 10⁻⁶ M.     

The identification of a domain in Escherichia coli elongation factor Tu that interacts with elongation factor Ts.

A method has been developed to search for the elongation factor Tu (EF-Tu) domain(s) that interact with elongation factor Ts (EF-Ts). This method is based on the suppression of Escherichia coli EF-Tu-dominant negative mutation K136E, a mutation that exerts its effect by sequestering EF-Ts. We have identified nine single-amino acid- substituted suppression mutations in the region 146-199 of EF-Tu. These mutations are R154C, P168L, A174V, K176E, D181G, E190K, D196G, S197F, and I199V. All suppression mutations but one (R154C) significantly affect EF-Tu's ability to interact with EF-Ts under equilibrium conditions. Moreover, with the exception of mutation A174V, the GDP affinity of EF-Tu appears to be relatively unaffected by these mutations. These results suggest that the domain of residues 154 to 199 on EF-Tu is involved in interacting with EF-Ts. These suppression mutations are also capable of suppressing dominant negative mutants N135D and N135I to various degrees. This suggests that dominant negative mutants N135D and N135I are likely to have the same molecular basis as the K136E mutation. The method we have developed in this study is versatile and can be readily adapted to map other regions of EF-Tu. A model of EF-Ts-catalyzed guanine-nucleotide exchange is discussed.     

Kinetic mechanism of elongation factor Ts-catalyzed nucleotide exchange in elongation factor Tu.

The interaction of Escherichia coli elongation factor Tu (EF-Tu) with elongation factor Ts (EF-Ts) and guanine nucleotides was studied by the stopped-flow technique, monitoring the fluorescence of tryptophan 184 in EF-Tu or of the mant group attached to the guanine nucleotide. Rate constants of all association and dissociation reactions among EF-Tu, EF-Ts, GDP, and GTP were determined. EF-Ts enhances the dissociation of GDP and GTP from EF-Tu by factors of 6 x 10(4) and 3 x 10(3), respectively. The loss of Mg(2+) alone, without EF-Ts, accounts for a 150-300-fold acceleration of GDP dissociation from EF-Tu.GDP, suggesting that the disruption of the Mg(2+) binding site alone does not explain the EF-Ts effect. Dissociation of EF-Ts from the ternary complexes with EF-Tu and GDP/GTP is 10(3)-10(4) times faster than from the binary complex EF-Tu.EF-Ts, indicating different structures and/or interactions of the factors in the binary and ternary complexes. Rate constants of EF-Ts binding to EF-Tu in the free or nucleotide-bound form or of GDP/GTP binding to the EF-Tu.EF-Ts complex range from 0.6 x 10(7) to 6 x 10(7) M(-1) s(-1). At in vivo concentrations of nucleotides and factors, the overall exchange rate, as calculated from the elemental rate constants, is 30 s(-1), which is compatible with the rate of protein synthesis in the cell.     

Qbeta-phage resistance by deletion of the coiled-coil motif in elongation factor Ts

Elongation factor Ts (EF-Ts) is the guanine-nucleotide exchange factor of elongation factor Tu (EF-Tu), which promotes the binding of aminoacyl-tRNA to the mRNA-programmed ribosome in prokaryotes. The EF-Tu.EF-Ts complex, one of the EF-Tu complexes during protein synthesis, is also a component of RNA-dependent RNA polymerases like the polymerase from coliphage Qbeta. The present study shows that the Escherichia coli mutant GRd.tsf lacking the coiled-coil motif of EF-Ts is completely resistant to phage Qbeta and that Qbeta-polymerase complex formation is not observed. GRd.tsf is the first E. coli mutant ever described that is unable to form a Qbeta-polymerase complex while still maintaining an almost normal growth behavior. The phage resistance correlates with an observed instability of the mutant EF-Tu.EF-Ts complex in the presence of guanine nucleotides. Thus, the mutant EF-Tu.EF-Ts is the first EF-Tu.EF-Ts complex ever described that is completely inactive in the Qbeta-polymerase complex despite its almost full activity in protein synthesis. We propose that the role of EF-Ts in the Qbeta-polymerase complex is to control and trap EF-Tu in a stable conformation with affinity for RNA templates while unable to bind aminoacyl-tRNA.     

Molecular characterization of the mitochondrial elongation factor EF-Tu gene in rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.)

The mitochondrial elongation factor EF-Tu (tufM) in rice (Oryza sativa L.) was isolated and characterized. The rice tufM cDNA clone contained 1,726 nucleotides and coded for a 453 amino acid protein including a putative mitochondrial transit peptide of 64 amino acid residues. This coding region was composed of 12 exons and 11 introns. The deduced amino acid sequence showed 62% and 88% identities with rice chloroplast EF-Tu (tufA) and Arabidopsis mitochondrial EF-Tu, respectively. As previously observed for the rice tufA gene, the tufM gene is likely present as one copy in rice. The mitochondrial EF-Tu gene was differentially expressed during flower development, and the other translational EF-Tu genes (chloroplast EF-Tu and cytosolic EF-1 alpha) were also distinctly expressed in a temporal manner. Phylogenetic analysis of the rice tufM gene showed that the mitochondrial tufA homologue of Reclinomonas was more closely related to the mitochondrial tufM genes of flowering plants than fungal and other mitochondrial tuf genes. In addition, the tufM encoded an N-terminal extension showing significant similarity to that of rps14 (or sdhB), which is also a nuclear-encoded rice mitochondrial gene.     

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.     

Cross-reactivity studies on the interaction between elongation factors Tu and Ts from Streptomyces aureofaciens and Escherichia coli in the GDP exchange reaction

The method of purification of elongation factor Ts from Streptomyces aureofaciens is described. Purified elongation factors Ts from S. aureofaciens and Escherichia coli were tested in cross-reactivity studies with elongation factors Tu from both species in a GDP exchange reaction under equilibrium and non-equilibrium conditions. Experiments have revealed that slower spontaneous release of GDP from S. aureofaciens EF-Tu is compensated for by higher affinity of homologous EF-Ts towards EF-Tu and thus the initial rates of EF-Ts catalysed GDP exchange can be kept the same in both E. coli and S. aurefaciens in vitro systems.     

Cloning and nucleotide sequence of the gene for an archaebacterial protein synthesis elongation factor Tu

Summary A restriction fragment enrichment procedure was devised for the identification and cloning of the gene for protein synthesis elongation factor Tu (EF-Tu) from Methanococcus vannielii, employing hybridisation with an internal tufB gene probe from Escherichia coli. Methanococcus contains a single tuf gene on its chromosome; it is expressed in E. coli and it codes for a polypeptide of 46.5 kDa. The overall architecture of the protein bears a striking resemblance to that of eukaryotic elongation factor 1 (EF-1). The close similarity to EF-1 is supported by the sequence homology values which are in the range of 34% to 35% with eubacterial, plastid and mitochondrial EF-Tu sequences and as high as 52% to 54% with those from eukaryotic EF-1.     

Intrinsic fluorescence of elongation factor Tu in its complexes with GDP and elongation factor Ts

The intrinsic fluorescence properties of elongation factor Tu (EF-Tu) in its complexes with GDP and elongation factor Ts (EF-Ts) have been investigated. The emission spectra for both complexes are dominated by the tyrosine contribution upon excitation at 280 nm whereas excitation at 300 nm leads to exclusive emission from the single tryptophan residue (Trp-184) of EF-Tu. The fluorescence lifetime of this tryptophan residue in both complexes was investigated by using a multifrequency phase fluorometer which achieves a broad range of modulation frequencies utilizing the harmonic content of a mode-locked laser. These results indicated a heterogeneous emission with major components near 4.8 ns for both complexes. Quenching experiments on both complexes indicated limited accessibility of the tryptophan residue to acrylamide and virtually no accessibility to iodide ion. The quenching patterns exhibited by EF-Tu-GDP and EF-Tu X EF-Ts were, however, different; both quenchers were more efficient at quenching the emission from the EF-Tu x EF-Ts complex. Steady-state and dynamic polarization measurements revealed limited local mobility for the tryptophan in the EF-Tu x GDP complex whereas formation of the EF-Tu x EF-Ts complex led to a dramatic increase in this local mobility.     

Isolation,expression, and evolution of the gene encoding mitochondrial elongation factor Tu in Arabidopsis thaliana

We have characterized a second nuclear gene (tufM) in Arabidopsis thaliana that encodes a eubacterial-like protein synthesis elongation factor Tu (EF-Tu). This gene does not closely resemble the previously described Arabidopsis nuclear tufA gene, which encodes the plastid EF-Tu, and does not contain sequence elements found in all cyanobacterial and plastid tufA genes. However, the predicted amino acid sequence includes an N-terminal extension which resembles an organellar targeting sequence and shares three unique sequence elements with mitochondrial EF-Tu's, from Saccharomyces cerevisiae and Homo sapiens, suggesting that this gene encodes the Arabidopsis mitochondrial EF-Tu. Consistent with this interpretation, the gene is expressed at a higher level in flowers than in leaves. Phylogenetic analysis confirms the mitochondrial character of the sequence and indicates that the human, yeast, and Arabidopsis tufM genes have undergone considerably more sequence divergence than their cytoplasmic counterparts, perhaps reflecting a cross-compartmental acceleration of gene evolution for components of the mitochondrial translation apparatus. As previously observed for tufA, the tufM gene is present in one copy in Arabidopsis but in several copies in other species of crucifers.