Site-directed mutagenesis of elongation factor Tu. The functional and structural role of residue Cys81.

A Cys residue located in the second consensus sequence element (DCPG) of the GTP-binding region is highly conserved in bacterial elongation factors (EF) Tu. Chemical modification of this Cys81 in EF-Tu from Escherichia coli by N-tosyl-L-phenylalanine chloromethane [Jonák, J., Petersen, T. E., Clark, B. F. C. & Rychlík, I. (1982) FEBS Lett. 150, 485-488], and of homologous Cys residues in other bacterial EF-Tu, selectively blocks the binding of Xaa-tRNA. We have substituted Cys81 with Gly using site-directed mutagenesis of the EF-Tu-encoding tuf A gene. This substitution induces a partial inhibition (20-70%) of: (a) poly(U)-directed poly(Phe) synthesis; (b) EF-Tu/Xaa-tRNA interaction, determined as protection by EF-Tu of the non-enzymic deacylation of Xaa-tRNA; (c) EF-Tu-dependent binding of Xaa-tRNA to the mRNA/ribosome complex and (d) the intrinsic GTPase reaction, that is also less sensitive to stimulation by Xaa-tRNA. Our results thus provide evidence that Cys81, though important, is not essential for the binding of Xaa-tRNA to EF-Tu. The accuracy in poly(Phe) synthesis, measured as misincorporation of Leu, was increased. Both the binding affinity of [C81G]EF-Tu for the nucleotide and the resistance against thermal denaturation are more strongly decreased in the case of the GDP-bound state than in the case of the GTP-bound state, suggesting that Cys81 plays a more specific role in the former conformation. The sensitivity to N-tosyl-L-phenylalanine chloromethane is decreased by 80% but not totally lost. The inhibition by N-tosyl-L-phenylalanine chloromethane treatment of the function of EF-Tu appears to be a consequence of steric hindrance and/or of an altered conformation of EF-Tu.GTP. The lower activities of [C81G]EF-Tu are probably due to long-range effects, mediated by an overall destabilization of the molecule that is particularly pronounced for the GDP-bound state.     

N-tosyl-L-phenylalanylchloromethane reacts with cysteine 81 in the molecule of elongation factor Tu from Escherichia coli

Elongation factor EF-Tu from Escherichia coli was labelled with N-[¹⁴C]tosyl-L-phenylalanylchloromethane, digested with trypsin and the peptides obtained separated by HPLC. The only radioactive peak recovered corresponded to tryptic peptide containing residues 75–98. Sequencing of the peptide by automated Edman degradation identified cysteine 81 as the site of N-tosyl-L-phenylalanylchloromethane modification. These results confirm the importance of this residue for the interaction with aminoacyl-tRNAs.     

Fluorescence characterization of the interaction of various transfer RNA species with elongation factor Tu.GTP: evidence for a new functional role for elongation factor Tu in protein biosynthesis

The ubiquity of elongation factor Tu (EF-Tu)-dependent conformational changes in amino-acyl-tRNA (aa-tRNA) and the origin of the binding energy associated with aa-tRNA.EF-Tu.GTP ternary complex formation have been examined spectroscopically. Fluorescein was attached covalently to the 4-thiouridine base at position 8 (s4U-8) in each of four elongator tRNAs (Ala, Met-m, Phe, and Val). Although the probes were chemically identical, their emission intensities in the free aa-tRNAs differed by nearly 3-fold, indicating that the dyes were in different environments and hence that the aa-tRNAs had different tertiary structures near s4U-8. Upon association with EF-Tu.GTP, the emission intensities increased by 244%, 57%, or 15% for three aa-tRNAs due to a change in tRNA conformation; the fourth aa-tRNA exhibited no fluorescence change upon binding to EF-Tu.GTP. Despite the great differences in the emission intensities of the free aa-tRNAs and in the magnitudes of their EF-Tu-dependent intensity increases, the emission intensity per aa-tRNA molecule was nearly the same (within 9% of the average) for the four aa-tRNAs when bound to EF-Tu-GTP. Thus, the binding of EF-Tu.GTP induced or selected a tRNA conformation near s4U-8 that was very similar, and possibly the same, for each aa-tRNA species. It therefore appears that EF-Tu functions, at least in part, by minimizing the conformational diversity in aa-tRNAs prior to their beginning the recognition and binding process at the single decoding site on the ribosome. Since an EF-Tu-dependent fluorescence change was also observed with fluorescein-labeled tRNA(Phe), the protein-dependent structural change is effected by direct interactions between EF-Tu and the tRNA and does not require the aminoacyl group. The Kd of the tRNA(Phe).EF-Tu.GTP ternary complex was determined, at equilibrium, to be 2.6 microM by the ability of the unacylated tRNA to compete with fluorescent Phe-tRNA for binding to the protein. Comparison of this Kd with that of the Phe-tRNA ternary complex showed that in this case the aminoacyl moiety contributed 4.3 kcal/mol toward ternary complex formation at 6 degrees C but that the bulk of the binding energy in the ternary complex was derived from direct protein-tRNA interactions.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.     

Interaction of mitochondrial elongation factor Tu with aminoacyl-tRNA and elongation factor Ts

Elongation factor (EF) Tu promotes the binding of aminoacyl-tRNA (aa-tRNA) to the acceptor site of the ribosome. This process requires the formation of a ternary complex (EF-Tu.GTP.aa-tRNA). EF-Tu is released from the ribosome as an EF-Tu.GDP complex. Exchange of GDP for GTP is carried out through the formation of a complex with EF-Ts (EF-Tu.Ts). Mammalian mitochondrial EF-Tu (EF-Tu(mt)) differs from the corresponding prokaryotic factors in having a much lower affinity for guanine nucleotides. To further understand the EF-Tu(mt) subcycle, the dissociation constants for the release of aa-tRNA from the ternary complex (K(tRNA)) and for the dissociation of the EF-Tu.Ts(mt) complex (K(Ts)) were investigated. The equilibrium dissociation constant for the ternary complex was 18 +/- 4 nm, which is close to that observed in the prokaryotic system. The kinetic dissociation rate constant for the ternary complex was 7.3 x 10(-)(4) s(-)(1), which is essentially equivalent to that observed for the ternary complex in Escherichia coli. The binding of EF-Tu(mt) to EF-Ts(mt) is mutually exclusive with the formation of the ternary complex. K(Ts) was determined by quantifying the effects of increasing concentrations of EF-Ts(mt) on the amount of ternary complex formed with EF-Tu(mt). The value obtained for K(Ts) (5.5 +/- 1.3 nm) is comparable to the value of K(tRNA).     

Purification of chloroplast elongation factor Tu and cDNA analysis in tobacco: the existence of two chloroplast elongation factor Tu species

We have purified a chloroplast elongation factor Tu (EF-Tu) from tobacco (Nicotiana tabacum) and determined its N-terminal amino acid sequence. Two distinct cDNAs encoding EF-Tu were isolated from a leaf cDNA library of N. sylvestris (the female progenitor of N. tabacum) using an oligonucleotide probe based on the EF-Tu protein sequence. The cDNA sequence and genomic Southern analyses revealed that tobacco chloroplast EF-Tu is encoded by two distinct genes in the nuclear genome of N. sylvestris. We designated the corresponding gene products EF-Tu A and B. The mature polypeptides of EF-Tu A and B are 408 amino acids long and share 95.3% amino acid identity. They show 75–78% amino acid identity with cyanobacterial and chloroplast-encoded EF-Tu species.     

Structural determination of the functional sites of E. coli elongation factor Tu

Recently, we have made significant progress in solving the structure of a nicked form of elongation factor (EF)-Tu complexed with GDP. The structure has been refined to an R factor of 19.2% at 2.6 A resolution, so that most of the structure is clearly visible in the electron density map. Here we describe what is known about functional sites of EF-Tu in terms of the structure, which still lacks amino acids 40-60.     

Crystals of partially trypsin-digested elongation factor Tu

Limited tryptic digestion of elongation factor Tu from Escherichia coli and Bacillus stearothermophilus at room temperature produces a small number of scissions without concomitant loss of GDP binding activity. The small number of large tryptic fragments produced are not separated by gel filtration under non-denaturing conditions and they coelute with the GDP binding activity. Crystals of the trypsin-treated elongation factor Tu from E. coli obtained from polyethylene glycol solutions are apparently identical to the pseudotetragonal crystals previously reported (Sneden et al., 1973).     

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.     

The busiest of all ribosomal assistants: elongation factor Tu

During translation, the nucleic acid language employed by genes is translated into the amino acid language used by proteins. The translator is the ribosome, while the dictionary employed is known as the genetic code. The genetic information is presented to the ribosome in the form of a mRNA, and tRNAs connect the two languages. Translation takes place in three steps: initiation, elongation, and termination. After a protein has been synthesized, the components of the translation apparatus are recycled. During each phase of translation, the ribosome collaborates with specific translation factors, which secure a proper balance between speed and fidelity. Notably, initiation, termination, and ribosomal recycling occur only once per protein produced during normal translation, while the elongation step is repeated a large number of times, corresponding to the number of amino acids constituting the protein of interest. In bacteria, elongation factor Tu plays a central role during the selection of the correct amino acids throughout the elongation phase of translation. Elongation factor Tu is the main subject of this review.     

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.     

Interaction of cinnamyl-tRNAPhe with Escherichia coli elongation factor Tu

The products of nitrous acid mediated-deamination of Phe-tRNAPhe from E. coli were analyzed and their capability to interact with elongation factor Tu from E. coli was investigated. Thin-layer chromatography as well as HPLC analysis revealed the existence of at least two deamination products, 3-phenyl-lactyl-tRNAPhe and cinnamyl-tRNAPhe. It could be shown that the aminoacyl-tRNA analogues were active in the formation of the ternary complex with EF-Tu X GTP, although with a lower efficiency than native Phe-tRNAPhe. For both modified acyl-tRNAs the dissociation constant was determined to be 3 X 10(-5) M.     

Binding of eucaryotic elongation factor Tu to nucleic acids

The binding of eucaryotic elongation factor Tu (eEF-Tu) to nucleic acids was investigated. eEF-Tu binds to a variety of different nucleic acids with high affinity, showing a strong preference for 18 S and 28 S rRNA over transfer RNA and for ribose-containing polymers over polydeoxyribonucleotides. The factor binds at multiple sites on 28 S rRNA without strong cooperativity. eEF-Tu binds strongly to poly(G) and poly(U) but weakly, if at all, to poly(A) and poly(C). Experiments employing an airfuge demonstrate that eEF-Tu can form a quaternary complex containing the factor, 28 S rRNA, aminoacyl-tRNA, and GTP. The existence of two distinct RNA binding sites on eEF-Tu suggests that rRNA may play a role in the recognition of eEF-Tu.aminoacyl-tRNA.GTP complexes by polysomes. Support for this suggestion comes from experiments which show that poly(G) inhibits the factor-dependent binding of aminoacyl-tRNA to mRNA-programmed 80 S ribosomes. In addition, it is shown that eEF-Tu possesses an intrinsic GTPase activity which is stimulated significantly by 28 S rRNA, poly(G), and poly(U). The binding of eEF-Tu to poly(G) lowers the activation energy for eEF-Tu GTPase from 74.3 to 65.9 kJ . mol-1 and approximately doubles the Vmax of the enzymatic reaction. The results are discussed in relation to the binding of eEF-Tu to ribosomes during protein synthesis.     

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.     

Chaperone properties of mammalian mitochondrial translation elongation factor Tu

The main function of the prokaryotic translation elongation factor Tu (EF-Tu) and its eukaryotic counterpart eEF1A is to deliver aminoacyl-tRNA to the A-site on the ribosome. In addition to this primary function, it has been reported that EF-Tu from various sources has chaperone activity. At present, little information is available about the chaperone activity of mitochondrial EF-Tu. In the present study, we have examined the chaperone function of mammalian mitochondrial EF-Tu (EF-Tumt). We demonstrate that recombinant EF-Tumt prevents thermal aggregation of proteins and enhances protein refolding in vitro and that this EF-Tumt chaperone activity proceeds in a GTP-independent manner. We also demonstrate that, under heat stress, the newly synthesized peptides from the mitochondrial ribosome specifically co-immunoprecipitate with EF-Tumt and are destabilized in EF-Tumt-overexpressing cells. We show that most of the EF-Tumt localizes on the mitochondrial inner membrane where most mitochondrial ribosomes are found. We discuss the possible role of EF-Tumt chaperone activity in protein quality control in mitochondria, with regard to the recently reported in vivo chaperone function of eEF1A.     

In vivo methylation of prokaryotic elongation factor Tu.

In Salmonella typhimurium and Escherichia coli, elongation factor Tu (EF-Tu) is methylated as shown by its incorporation of labeled methyl residues from [methyl-3H]methionine. Analysis of the nature of the methyl-containing residues by protein hydrolysis, followed by paper chromatography and high voltage electrophoresis showed that both mono- and dimethyllysine are present. Eighty per cent of the EF-Tu molecules are methylated if methylation occurs at a unique lysine residue. The EF-Tu fraction which is not methylated is still able to accept methyl groups, as shown by methylation of approximately 10% of the EF-Tu after addition of chloramphenicol (D-(-)-threo-2,2-dichloro-N-[beta-hydroxy-alpha-(hydroxymethyl)-o-nitrophenethyl] acetamide) to inhibit further protein synthesis. There is no evidence of turnover of the methyl residues. We attempted to separate the methylated from the nonmethylated form of EF-Tu by isoelectric focusing on polyacrylamide gel, but were unable to do so.