Structural fluctuation of the polypeptide-chain elongation factor Tu. A comparison of factors from Escherichia coli and Thermus thermophilus HB8.

The kinetics of hydrogen-deuterium exhcange in the polypeptide chain elongation factor Tu (EF Tu) from Escherichia coli and that from Thermus thermophilus HB8 has been examined in aqueous solutions at various pH and temperatures by means of infrared absorption measurements. The free EF-Tu from E. Coli has a greater reaction rate at all pH values and at every temperature than that of the GTP-bound or GDP-bound EF-Tu. The free EF-Tu from T. thermophilus, on the other hand, has an alomst equal reaction rate to that of EF-Tu-GDP in the temperature range 38-55 degrees C. For the peptide NH groups belonging to a medium-labile kinetic class, a small but definite difference in the rate of exchange reaction was observed between EF-Tu-GDP and EF-Tu-GTP for both E. coli and T. thermophilus. For less labile peptide NH groups, on the other hand, the rate of the exchange reaction with EF-Tu-GDP from T. thermophilus is only slightly affected by the pH of the solution at 38 degrees C and 45 degrees C, while the rate constant(k) with E. coli EF-Tu-GDP is pH-dependent (log k oc pH). For T. thermophilus EF-Tu, heat stability measurements, kinetics of the rates of GDP and GTP dissociation, and circular dichroic measurements have also been made. The molecular basis for the thermostability of T. thermophilus EF-Tu is discussed.     

Sequence and identification of the nucleotide binding site for the elongation factor Tu from Thermus thermophilus HB8.

Two structural genes for the Thermus thermophilus elongation factor Tu (tuf) were identified by cross-hybridization with the tufA gene from E. coli. The sequence of one of these tuf genes, localized on a 6.6 kb Bam HI fragment, was determined and confirmed by partial protein sequencing of an authentic elongation factor Tu from T. thermophilus HB8. Expression of this tuf gene in E. coli minicells provided a low amount of immuno-precipitable thermophilic EF-Tu. Affinity labeling of the T. thermophilus EF-Tu and sequence comparison with homologous proteins from other organisms were used to identify the guanosine-nucleotide binding domain.     

Studies on polypeptide-chain-elongation factors from an extreme thermophile, Thermus thermophilus HB8. 2. Catalytic properties.

Catalytic properties of the elongation factors from Thermus thermophilus HB8 have been studied and compared with those of the factors from Escherichia coli. 1. The formation of a ternary guanine-nucleotide . EF-Tu . EF-Ts complex was demonstrated by gel filtration of the T. thermophilus EF-Tu . EF-Ts complex on a Sephadex G-150 column equilibrated with guanine nucleotide. The occurrence of this type of complex has not yet been proved with the factors from E. coli. 2. The dissociation constants for the complexes of T. thermophilus EF-Tu . EF-Ts with GDP and GTP were 6.1 x 10(-7) M and 1.9 x 10(-6) M respectively. On the other hand, T. thermophilus EF-Tu interacted with GDP and GTP with dissociation constants of 1.1 x 10(-9) M and 5.8 x 10(-8) M respectively. This suggests that the association of EF-Ts with EF-Tu lowered the affinity of EF-Tu for GDP by a factor of about 600 and facilitated the nucleotide exchange reaction. 3. Although the T. thermophilus EF-Tu . EF-Ts complex hardly dissociates into EF-Tu and EF-Ts, a rapid exchange was observed between free EF-Ts and the EF-Tu . EF-Ts complex using 3H-labelled EF-Ts. The exchange reaction was independent on the presence or absence of guanine nucleotides. 4. Based on the above findings, an improved reaction mechanism for the regeneration of EF-Tu . GTP from EF-Tu . GDP is proposed. 5. Studies on the functional interchangeability of EF-Tu and EF-Ts between T. thermophilus and E. coli has revealed that the factors function much more efficiently in the homologous than in the heterologous combination. 6. T. thermophilus EF-Ts could bind E. coli EF-Tu to form an EF-Tu (E. coli) . EF-Ts (T. thermophilus hybrid complex. The complex was found to exist in a dimeric form indicating that the property to form a dimer is attributable to T. thermophilus EF-Ts. On the other hand, no stable complex between E. coli EF-Ts and T. thermophilus EF-Tu has been isolated. 7. The uncoupled GTPase activity of T. thermophilus EF-G was much lower than that of E. coli EF-G. T. thermophilus EF-G formed a relatively stable binary EF-G . GDP complex, which could be isolated on a nitrocellulose membrane filter. The Kd values for EF-G . GDP and EF-G . GTP were 6.7 x 10(-7) M and 1.2 x 10(-5) M respectively. The ternary T. thermophilus EF-G . GDP . ribosome complex was again very stable and could be isolated in the absence of fusidic acid. The stability of the latter complex is probably the cause of the low uncoupled GTPase activity of T. thermophilus EF-G.     

Studies on polypeptide-chain-elongation factors from an extreme thermophile, Thermus thermophilus HB8. 3. Molecular properties.

Molecular properties of the polypeptide chain elongation factors from Thermus thermophilus HB8 have been investigated and compared with those from Escherichia coli. 1. As expected, the factors purified from T. thermophilus were exceedingly heat-stable. Even free EF-Tu not complexed with GDP was stable after heating for 5 min at 60 degrees C. 2. GDP binding activity of T. thermophilus EF-Tu was also stable in various protein denaturants, such as 5.5 M urea, 1.5 M guanidine-HCl, and 4 M LiCl. 3. Amino acid compositions of EF-Tu and EF-G from T. thermophilus were similar to those from E. coli. On the other hand, amino acid composition of T. thermophilus EF-Ts was considerably different from that of E. coli EF-Ts. 4. In contrast to E. coli EF-Tu, T. thermophilus EF-Tu contained no free sulfhydryl group, but one disulfide bond. The disulfide bond was cleaved by sodium borohydride or sodium sulfite under native conditions. The heat stability of the reduced EF-Tu . GDP, as measured by GDP binding activity, did not differ from that of the untreated EF-Tu . GDP. 5. T. thermophilus EF-Ts contained, in addition to one disulfide bond, a sulfhydryl group which could be titrated only after complete denaturation of the protein. 6. Under native conditions one sulfhydryl group of T. thermophilus EF-G was titrated with p-chloromercuribenzoate, while the rate of reaction was very sluggish. The sulfhydryl group appears to be essential for interaction with ribosomes, whereas the ability to form a binary GDP . EF-G complex was not affected by its modification. The protein contained also one disulfide bond. 7. Circular dichroic spectra of EF-Tu from T. thermophilus and E. coli were very similar. Binding of GDP or GTP caused a similar spectral change in both. T. thermophilus and E. coli EF-Tu. On the other hand, the spectra of T. thermophilus EF-G and E. coli EF-G were significantly different, the content of ordered structure being higher in the former as compared to the latter.     

Molecular cloning and sequence determination of the tuf gene coding for the elongation factor Tu of Thermus thermophilus HB8

The tuf gene, which encodes the elongation factor Tu (EF-Tu) of Thermus thermophilus HB8, and its flanking regions were cloned and sequenced. The gene encoding EF-G was found upstream of the 5' end of the tuf gene. The tuf gene of T. thermophilus HB8 had a very high G + C content and 84.5% of the third base in codon usage was either G or C. The deduced primary structure of the EF-Tu was composed of 405 amino acid residues with a Mr = 44658. A comparison of the amino acid sequence of EF-Tu from T. thermophilus HB8 with those of Escherichia coli and Saccharomyces cerevisiae mitochondria showed a very high sequence homology (65-70%). Two Cys residues out of the three found in E. coli EF-Tu had been replaced with Val in T. thermophilus HB8 EF-Tu. An extra amino acid sequence of ten residues, consisting predominantly of basic amino acids (Met-182-Gly-191), which does not occur in EF-Tu of E. coli, was found in T. thermophilus HB8.     

Affinity labeling of the GDP/GTP binding site in Thermus thermophilus elongation factor Tu

Elongation factor Tu from Thermus thermophilus was treated successively with periodate-oxidized GDP or GTP and cyanoborohydride. Covalently modified cyanogen bromide or trypsin fragments of the protein were isolated, and the position of their modification was determined. Lysine residues 52 and 137 were heavily labeled, lysine-137 being considerably more reactive in the GTP form as compared to the GDP form of the protein. These residues are in the proximity of the GDP/GTP binding site. Lys-325 was also labeled, but to a lower extent. The part of the EF-Tu containing residue 52 is missing in crystallized EF-Tu.GDP from Escherichia coli [Jurnak, F. (1985) Science (Washington, D.C.) 230, 32-36]. These results place the part of T. thermophilus EF-Tu corresponding to the missing fragment in E. coli EF-Tu in the vicinity of the nucleotide binding site and allow its role in the interaction with aminoacyl-tRNA and elongation factor Ts to be evaluated. Cross-linking of EF-Tu.GDP by irradiation at 257 nm showed that a sequence of 10 amino acids residues which is found in the Thermus thermophilus elongation factor Tu but not in other homologous bacterial proteins is located in the vicinity of the GDP/GTP binding site.     

Genetic and molecular analysis of the tRNA-tufB operon of the myxobacterium Stigmatella aurantiaca.

The tufB gene, encoding elongation factor Tu (EF-Tu), from the myxobacterium Stigmatella aurantiaca was cloned and sequenced. It is preceded by four tRNA genes, the first ever described in myxobacteria. The tRNA synthesized from these genes and the general organization of the locus seem identical to that of Escherichia coli, but differences of potential importance were found in the tRNA sequences and in the intergenic regions. The primary structure of EF-Tu was deduced from the tufB DNA sequence. The factor is composed of 396 amino acids, with a predicted molecular mass of 43.4 kDa, which was confirmed by expression of tufB in maxicells. Sequence comparisons between S.aurantiaca EF-Tu and other bacterial homologues from E.coli, Salmonella typhimurium and Thermus thermophilus displayed extensive homologies (75.9%). Among the variable positions, two Cys residues probably involved in the temperature sensitivity of E.coli and S.typhimurium EF-Tu are replaced in T.thermophilus and S.aurantiaca EF-Tu. Since two or even three tuf genes have been described in other bacterial species, the presence of multiple tuf genes was sought for. Southern and Northern analysis are consistent with two tuf genes in the genome of S.aurantiaca. Primer extension experiments indicate that the four tRNA genes and tufB are organized in a single operon.     

Sequence of the tufA gene encoding elongation factor EF-Tu from Thermus aquaticus and overproduction of the protein in Escherichia coli.

The sequence of the tufA gene from the extreme thermophilic eubacterium Thermus aquaticus EP 00276 was determined. The GC content in third positions of codons is 89.5%, with an unusual predominance of guanosine (60.7%). The derived protein sequence differs from tufA- and tufB-encoded sequences for elongation factor Tu (EF-Tu) of Thermus thermophilus HB8, another member of the genus Thermus, in 10 of the 405 amino acid residues. Three exchanges are located in the additional loop of ten amino acids (182-191). The loop, probably involved in nucleotide binding, is absent in EF-Tu of the mesophile Escherichia coli. Since EF-Tu from E. coli is quite unstable, the protein is well-suited for analyzing molecular changes that lead to thermostabilization. Comparison of the EF-Tu domain I from E. coli and Thermus strains revealed clustered amino acid exchanges in the C-terminal part of the first helix and in adjacent residues of the second loop inferred to interact with the ribosome. Most other exchanges in the guanine nucleotide binding domain are located in loops or nearest vicinity of loops suggesting their importance for thermostability. The T. aquaticus EF-Tu was overproduced in E. coli using the tac expression system. Identity of the recombinant T. aquaticus EF-Tu was verified by Western blot analysis, N-terminal sequencing and GDP binding assays.     

Molecular cloning, nucleotide sequence and expression of the tufB gene encoding elongation factor Tu from Thermus thermophilus HB8

The tufB gene encoding elongation factor Tu (EF-Tu) of Thermus thermophilus HB8 was cloned and expressed. Compared with the known tufA gene of T. thermophilus, nucleotide differences were found at 10 positions out of 1221 nucleotides, and amino acid substitutions were found at 4 positions out of 406 amino acids. The tufB product was 70.9% homologous to the corresponding sequence of the tufB product of E. coli. The G+C content of the third base of the codon in the tufB gene was 84.8% and G was especially preferred in this position.     

Pulvomycin-resistant mutants of E.coli elongation factor Tu.

This paper reports the generation of Escherichia coli mutants resistant to pulvomycin. Together with targeted mutagenesis of the tufA gene, conditions were found to overcome membrane impermeability, thereby allowing the selection of three mutants harbouring elongation factor (EF)-Tu Arg230-->Cys, Arg333-->Cys or Thr334-->Ala which confer pulvomycin resistance. These mutations are clustered in the three-domain junction interface of the crystal structure of the GTP form of Thermus thermophilus EF-Tu. This result shares similarities with kirromycin resistance; kirromycin-resistant mutations cluster in the domain 1-3 interface. Since both interface regions are involved in the EF-Tu switch mechanism, we propose that pulvomycin and kirromycin both act by specifically disturbing the allosteric changes required for the switch from EF-Tu-GTP to EF-Tu-GDP. The three-domain junction changes dramatically in the switch to EF-Tu.GDP; in EF-Tu.GDP this region forms an open hole. Structural analysis of the mutation positions in EF-Tu.GTP indicated that the two most highly resistant mutants, R230C and R333C, are part of an electrostatic network involving numerous residues. All three mutations appear to destabilize the EF-Tu.GTP conformation. Genetic and protein characterizations show that sensitivity to pulvomycin is dominant over resistance. This appears to contradict the currently accepted model of protein synthesis inhibition by pulvomycin.     

Properties of the elongation factor 1 alpha in the thermoacidophilic archaebacterium Sulfolobus solfataricus

The elongation factor 1 alpha (aEF-1 alpha) was purified to homogeneity from the thermoacidophilic archaebacterium Sulfolobus solfataricus by chromatographic procedures utilising DEAE-Sepharose, hydroxyapatite and FPLC on Mono S. The purified protein binds [3H]GDP at a 1:1 molar ratio and it is essential for poly(Phe) synthesis in vitro; it also binds GTP but not ATP. These findings indicate that aEF-1 alpha is the counterpart of the eubacterial elongation factor Tu (EF-Tu). Purified aEF-1 alpha is a monomeric protein with a relative molecular mass of 49,000 as determined by SDS/PAGE and by gel filtration on Sephadex G-100; its isoelectric point is 9.1. The overall amino acid composition did not reveal significant differences when compared with the amino acid composition of eubacterial EF-Tu from either Escherichia coli or Thermus thermophilus, of eukaryotic EF-1 alpha from Artemia salina or of archaebacterial EF-1 alpha from Methanococcus vannielii. The close similarities between the average hydrophobicity and the numbers of hydrogen-bond-forming or non-helix-forming residues suggest that common structural features exist among the factors compared. aEF-1 alpha shows remarkable thermophilic properties, as demonstrated by the rate of [3H]GDP binding which increases with temperature, reaching a maximum at 95 degrees C; it is also quite heat-resistant, since after a 6-h exposure at 60 degrees C and 87 degrees C the residual [3H]GDP-binding ability was still 90% and 54% of the control, respectively. The affinity of aEF-1 alpha for GDP and GTP was also evaluated. At 80 degrees C Ka' for GDP was about 30-fold higher than Ka' for GTP; at the same temperature Kd' for GDP was 1.7 microM and Kd' for GTP was 50 microM; these values were 300-fold and 100-fold higher, respectively, than those reported for E. coli EF-Tu at 30 degrees C; compared to the values at 0 degree C of EF-Tu from E. coli and T. thermophilus or EF-1 alpha from A. salina, pig liver and calf brain, smaller differences were observed with eukaryotic factors.(ABSTRACT TRUNCATED AT 400 WORDS)     

Conformational transitioons of polypeptide chain elongation factor Tu. II. Further studies by electron spin resonance.

The conformational transitions of polypeptide chain elongation factor Tu (EF-Tu) associated with the ligand change from GDP to GTP and also with the displacement of GDP by elongation factor Ts (EF-Ts) have been investigated using the spin-labeling technique. Of the two reactive sulfhydryl groups in EF-Tu, the one essential for interaction with aminoacyl-tRNA was selectively labeled with various kinds of iodoacetamide or maleimide spin-labeling reagents. The electron spin resonance (ESR) spectra of EF-Tu-GDP labeled with these reagents generally consisted of two components, one narrow and one broad, corresponding to labels relatively weakly and strongly immobilized, respectively. The degree of immobilization and the ratio of the narrow to the broad components were different for each kind of label used. The spectra of spin-labeled EF-Tu-GDP changed markedly when its GDP moiety was replaced by GTP through incubation with phosphoenolpyruvate and pyruvate kinase [EC 2.7.1.40], the broad component increasing at the expense of the narrow component. The reversible nature of the conformational change was confirmed with EF-Tu labeled with a maleimide reagent. The GTP-induced spectral change was reversed upon conversion of labeled EF-Tu-GTP to EF-Tu-GDP by addition of excess GDP. A similar type of spectral change was also observed when spin-labeled EF-Tu-GDP was incubated with EF-Ts to form labeled EF-Tu-EF-Ts complex. The extent of the spectral change induced by EF-Ts was even greater than that induced by GTP. These results, together with those obtained by studies with hydrophobic and fluorescent probes (Arai, Arai, Kawakita, & Kaziro (1975) J. Biochem. 77, 1095-1106) indicate that a reversible conformational change is induced in EF-Tu near the sulfhydryl group that is essential for interaction with aminoacyl-tRNA.     

Three-dimensional models of the GDP and GTP forms of the guanine nucleotide domain of Escherichia coli elongation factor Tu

Three-dimensional models of the GDP and GTP forms of the guanine nucleotide domain of Escherichia coli elongation factor Tu have been derived from the atomic coordinates of the trypsin-modified form of EF-Tu-GDP and by comparison with the ras p21 structures. The significance of the differences in the guanine nucleotide binding sites of EF-Tu and ras p21 are discussed. Crystallization of the EF-Tu-GMPPNP complex is reported.     

Spin-labelled analogues of GDP and GTP as site-specific reporter groups for guanosine nucleotide-binding proteins

New derivatives of GDP and GTP have been synthesized for the spectroscopic investigation of the interaction between guanosine nucleotides and guanosine nucleotide-binding proteins. The 3'-hydroxyl group in these nucleotides was replaced by a 3'-amino group, which was further derivatized by the introduction of a spin-label reporter group. The biological activity of 3'SL-GDP and 3'SL-GTP could be demonstrated by measuring the interaction of these spin-labelled derivatives with bacterial elongation factor Tu. The amino modification and spin labelling only slightly influenced the affinity of the guanosine nucleotides for EF-Tu from Escherichia coli or Thermus thermophilus. Electron paramagnetic resonance (EPR) measurements revealed a strong immobilization of the labelled nucleotides upon binding to T. thermophilus EF-Tu. Significant differences between the spectra of EF-Tu X 3'SL-GDP, EF-Tu X 3'SL-GTP and aminoacyl-tRNA X EF-Tu X 3'SL-GTP ternary complexes were observed. Our data demonstrate that spin-labelled guanosine nucleotides can be used as sensitive spectroscopic probes for the investigation of the local environment of the nucleotide-binding site during distinct functional states of a guanosine nucleotide-binding protein.     

Affinity labeling of GTP-binding proteins in cellular extracts.

GTP-binding proteins in cellular extracts from Escherichia coli, Thermus thermophilus, yeast, wheat germ or calf thymus were identified using in situ periodate-oxidized [alpha-32P]GTP as affinity label. Site-specific reaction of individual GTP-binding proteins was achieved by cross-linking the protein-bound 2',3'-dialdehyde derivative of GTP with the single lysine residue of the conserved NKXD sequence through Schiff's base formation and subsequent cyanoborohydride reduction. Labeled GTP-binding proteins from prokaryotic or eukaryotic cell homogenates were separated by polyacrylamide gel electrophoresis and visualized by autoradiography. In addition cross-linking of [alpha-32P]GTP with GTP-binding proteins was demonstrated in model systems using different purified GTPases, human c-H-ras p21, transducin from bovine retina, polypeptide elongation factor Tu (EF-Tu) from T. thermophilus and initiation factor 2 (IF2) from T. thermophilus. The described affinity labeling technique can serve as an analytical method for the identification of GTPases belonging to the classes of ras-proteins, elongation and initiation factors, and heterotrimeric signal transducing G-proteins.     

Psychrophilic elongation factor Tu from the antarctic Moraxella sp. Tac II 25: biochemical characterization and cloning of the encoding gene

The elongation factor Tu was isolated from a psychrophilic eubacterial Antarctic Moraxella strain (MoEF-Tu) and its molecular and functional properties were determined. It catalyzed the synthesis of poly(Phe) and bound specifically guanine nucleotides with an affinity for GDP about 12-fold higher than that for GTP. The affinity toward guanine nucleotides was lower than that of other eubacterial EF-Tu. The intrinsic GTPase activity of MoEF-Tu was hardly detectable but was accelerated by 2 orders of magnitude in the presence of the antibiotic kirromycin (GTPase(k)). Such a property resembled Escherichia coli EF-Tu (EcEF-Tu) even though the affinity of MoEF-Tu for the antibiotic was lower. MoEF-Tu showed a thermophilicity higher than that of EcEF-Tu; its temperature for half-denaturation was 44 degrees C. The MoEF-Tu encoding gene corresponding to E. coli tufA was cloned and sequenced. The translated protein had a calculated molecular weight of 43 288 and contained the GTP-binding sequence motifs. Concerning its primary structure, MoEF-Tu showed sequence identity with E. coli and Thermus thermophilus EF-Tu equal to 84% and 74%, respectively, while the identity with EF-1 alpha from the archaeon Sulfolobus solfataricus was equal to 32%.     

Purification and characterization of Saccharomyces cerevisiae mitochondrial elongation factor Tu

Yeast mitochondrial elongation factor Tu (EF-Tu) was purified 200-fold from a mitochondrial extract of Saccharomyces cerevisiae to yield a single polypeptide of Mr = approximately 47,000. The factor was detected by complementation with Escherichia coli elongation factor G and ribosomes in an in vitro phenylalanine polymerization reaction. Mitochondrial EF-Tu, like E. coli EF-Tu, catalyzes the binding of aminoacyl-tRNA to ribosomes and possesses an intrinsic GTP hydrolyzing activity which can be activated either by kirromycin or by ribosomes. Kinetic and binding analyses of the interactions of mitochondrial EF-Tu with guanine nucleotides yielded affinity constants for GTP and GDP of approximately 5 and 25 microM, respectively. The corresponding affinity constants for the E. coli factor are approximately 0.3 and 0.003 microM, respectively. In keeping with these observations, we found that purified mitochondrial EF-Tu, unlike E. coli EF-Tu, does not contain endogenously bound nucleotide and is not stabilized by GDP. In addition, we have been unable to detect a functional counterpart to E. coli EF-Ts in extracts of yeast mitochondria and E. coli EF-Ts did not detectably stimulate amino acid polymerization with mitochondrial EF-Tu or enhance the binding of guanine nucleotides to the factor. We conclude that while yeast mitochondrial EF-Tu is functionally analogous to and interchangeable with E. coli EF-Tu, its affinity for guanine nucleotides and interaction with EF-Ts are quite different from those of E. coli EF-Tu.     

Structural homology between elongation factors EF-Tu from Bacillus stearothermophilus and Escherichia coli in the binding site for aminoacyl-tRNA

Elongation factor EF-Tu (Mr approximately equal to 50 000) and elongation factor EF-G (Mr approximately equal to 78 000) were isolated from Bacillus stearothermophilus in a homogeneous form. The ability of EF-Tu to participate in protein synthesis is rapidly inactivated by N-tosyl-L-phenyl-alanylchloromethane (Tos-PheCH2Cl). EF-Tu X GTP is more susceptible to the inhibition by Tos-PheCH2Cl than is EF-Tu X GDP. Tos-PheCH2Cl forms a covalent equimolar complex with the factor by reacting with a cysteine residue in its molecule. The labelling of EF-Tu by the reagent irreversibly destroys its ability to bind aminoacyl-tRNA, which in turn protects the protein from this inactivation. This indicates that the modification of EF-Tu by Tos-PheCH2Cl occurs at the aminoacyl-tRNA binding site of the protein. To identify and characterize the site of aminoacyl-tRNA binding in EF-Tu, the factor was labelled with [14C]Tos-PheCH2Cl, digested with trypsin, the resulting peptides were separated by high-performance liquid chromatography and the sequence of the radioactive peptide was determined. The peptide has identical structure with an Escherichia coli EF-Tu tryptic peptide comprising the residues 75-89 and the Tos-PheCH2Cl-reactive cysteine at position 81 [Jonák, J., Petersen, T. E., Clark, B. F. C. and Rychlík, I. (1982) FEBS Lett. 150, 485-488]. Experiments on photo-oxidation of EF-Tu by visible light in the presence of rose bengal dye showed that there are apparently two histidine residues in elongation factor Tu from B. stearothermophilus which are essential for the interaction with aminoacyl-tRNA. This is clearly reminiscent of a similar situation in E. coli EF-Tu [Jonák, J., Petersen, T. E., Meloun, B. and Rychlík, I. (1984) Eur. J. Biochem. 144, 295-303]. Our results provide further evidence for the conserved nature of the site of aminoacyl-tRNA binding in elongation factor EF-Tu and show that Tos-PheCH2Cl reagent might be a favourable tool for the identification of the site in the structure of prokaryotic EF-Tus.     

Cloning, sequencing, and expression in Escherichia coli of the gene encoding a 45-kilodalton protein, elongation factor Tu, from Chlamydia trachomatis serovar F.

The gene encoding a 45-kDa protein (45K) of Chlamydia trachomatis serovar F was cloned, sequenced, and overexpressed in Escherichia coli. Alignment of the deduced peptide sequence with E. coli elongation factor Tu (EF-Tu) demonstrated 69% identity. The 45K was recognized by a Chlamydia genus-specific monoclonal antibody GP-45 and cross-reacted with a monospecific polyclonal antibody to E. coli EF-Tu. Purified recombinant 45K has the capability to bind GDP, and the binding was enhanced in the presence of E. coli elongation factor Ts (EF-Ts). The GDP binding was specifically inhibited by the monoclonal antibody GP-45. These data suggest that the 45K is a chlamydial EF-Tu, and it forms a functional complex with E. coli EF-Ts protein.