Structure-function relationships of elongation factor Tu as studied by mutagenesis.

We have modified elongation factor Tu (EF-Tu) from Escherichia coli via mutagenesis of its encoding tufA gene to study its function-structure relationships. The isolation of the N-terminal half molecule of EF-Tu (G domain) has facilitated the analysis of the basic EF-Tu activities, since the G domain binds the substrate GTP/GDP, catalyzes the GTP hydrolysis and is not exposed to the allosteric constraints of the intact molecule. So far, the best studied region has been the guanine nucleotide-binding pocket defined by the consensus elements typical for the GTP-binding proteins. In this area most substitutions were carried out in the G domain and were found to influence GTP hydrolysis. In particular, the mutation VG20 (in both G domain and EF-Tu) decreases this activity and enhances the GDP to GTP exchange; PT82 induces autophosphorylation of Thr82 and HG84 strongly affects the GTPase without altering the interaction with the substrate. SD173, a residue interacting with (O)6 of the guanine, abolishes the GTP and GDP binding activity. Substitution of residues Gln114 and Glu117, located in the proximity of the GTP binding pocket, influences respectively the GTPase and the stability of the G domain, whereas the double replacement VD88/LK121, located on alpha-helices bordering the GTP-binding pocket, moderately reduces the stability of the G domain without greatly affecting GTPase and interaction with GTP(GDP). Concerning the effect of ligands, EF-TuVG20 supports a lower poly(Phe) synthesis but is more accurate than wild-type EF-Tu, probably due to a longer pausing on the ribosome.(ABSTRACT TRUNCATED AT 250 WORDS)     

tRNA and the guanosinetriphosphatase activity of elongation factor Tu

Different sites of the tRNA molecule influence the activity of the elongation factor Tu (EF-Tu) center for GTP hydrolysis [Parlato, G., Pizzano, R., Picone, D., Guesnet, J., Fasano, O., & Parmeggiani, A. (1983) J. Biol. Chem. 258, 995-1000]. Continuing these studies, we have investigated some aspects of (a) the effect of different tRNA(Phe) species, including Ac-Phe-tRNA(Phe) and 3'-truncated tRNA-CCA in the presence and absence of codon-anticodon interaction, and (b) the effect of occupation of the ribosomal P-site by different tRNA(Phe) species. Surprisingly, we have found that 3'-truncated tRNA can enhance the GTPase activity in the presence of poly(U), in contrast to its inhibitory effect in the absence of codon-anticodon interaction. Moreover, Ac-Phe-tRNA(Phe) was found to have some stimulatory effect on the ribosome EF-Tu GTPase in the presence of poly(U). These results indicate that under specific conditions the 3'-terminal end and a free terminal alpha-NH2 group are not essential for the stimulation of the catalytic center of EF-Tu; therefore, the same structure of the tRNA molecule can act as a stimulator or an inhibitor of EF-Tu functions, depending on the presence of codon-anticodon interaction and on the concentration of monovalent and divalent cations. EF-Tu-GTP does not recognize a free ribosomal P-site from a P-site occupied by the different tRNA(Phe) species. When EF-Tu acts as a component of the ternary complex formed with GTP and aa-tRNA, the presence of tRNA in the P-site strongly increases the GTPase activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mutagenesis of the NH2-terminal domain of elongation factor Tu

Mutagenesis was carried out in the N-terminal domain of elongation factor Tu (EF-Tu) to characterize the structure-function relationships of this model GTP binding protein with respect to stability, the interaction with GTP and GDP, and the catalytic activity. The substitutions were introduced in elements around the guanine nucleotide binding site or in the loops defining this site, in the intact molecule or in the isolated N-terminal domain (G domain). The double substitution Val88----Asp and Leu121----Lys, two residues situated on two vicinal alpha-helices, influences the basic activities of the truncated factor to a limited extent, probably via long-range interactions, and induces a destabilisation of the G domain structure. The functional alterations brought about by substitutions on the consensus sequences 18-24 and 80-83 highlight the importance of these residues for the interaction with GTP/GDP and the GTPase activity. Mutations concerning residues interacting with the guanine base lead to proteins in large part insoluble and inactive. In one case, the mutated protein (EF-TuAsn135----Asp) inhibited the growth of the host cell. This demonstrates the crucial role of the base specificity for the active conformation of EF-Tu. The obtained results are discussed in the light of the three-dimensional structure of EF-Tu.     

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.     

Site-directed mutagenesis of the GDP binding domain of bacterial elongation factor Tu

The tertiary structure model of EF-Tu predicts that the amino acid sequence Val-Asp-His-Gly-Lys-Thr-Thr-Leu (residues 20-27) forms a pocket that binds the pyrophosphate group. To test this model we used site-directed mutagenesis to produce forms of EF-Tu altered in this region. The following mutations were constructed: Gly-20, Val-23, Glu-24, Ile-25, and Pro-27. Each protein was labeled with [35S]Met and was tested for its ability to interact with guanosine nucleotides and EF-Ts. The in vivo activity of each altered protein was tested by determining its ability to confer aurodox sensitivity to a resistant host. Mutations at residues 23, 24, 25, and 27 eliminated the ability of EF-Tu to interact with either guanosine nucleotides or EF-Ts in vitro, and these forms were also inactive in vivo. In contrast, the Gly-20 form was nearly as active as wild-type EF-Tu in vitro and in vivo. This mutation is theoretically equivalent to reversion of the Gly to Val transforming mutation of the cellular form of the ras gene product p21, a protein proposed to be structurally similar to EF-Tu in the GDP binding domain. In contrast to its effect in the ras gene, the Val to Gly conversion did not affect the endogenous GTPase of EF-Tu. We conclude that the tertiary structure model is correct in its assignment of the pyrophosphate binding site to residues 23-27; however, there are likely to be some significant differences between the configurations of the GTPases of EF-Tu and p21.     

Substitution of proline 82 by threonine induces autophosphorylating activity in GTP-binding domain of elongation factor Tu

Mutation of Pro82 into Thr, a residue situated in the second element (D80CPG83) of the consensus sequence proposed to interact with GTP/GDP in GTP-binding proteins was introduced via site-directed mutagenesis in the isolated guanine nucleotide-binding domain (G domain) of elongation factor Tu. G domainPT82 displays virtually no GTPase activity. As a major change, the apparent inhibition of the GTPase reaction is associated with the appearance of autophosphorylating activity, as in ras product p21 in the case of mutation Ala59----Thr, corresponding to 82 in elongation factor Tu. Dependence of this reaction on mono- and divalent cation concentration and on pH is essentially the same as for the GTPase of wild-type G domain. The autokinase reaction follows an apparent first order rate, suggesting an intermolecular mechanism. Analysis of amino acid and peptide composition of the 32P-labeled G domainPT82, as well as Edman degradation of the tryptic peptide containing the covalently bound 32P, shows that Thr82 is the phosphorylated residue. Taken together, these results point out that Thr82 is in close proximity to the gamma-phosphate of GTP, as in the case of Thr59 in p21. These results are in agreement with the observations derived from x-ray diffraction analysis that the tertiary structure of the GTP-binding domain of elongation factor Tu and that of p21 are similar.     

Isolation and stability of ternary complexes of elongation factor Tu, GTP and aminoacyl-tRNA.

Intact, native EF-Tu, isolated using previously described methods and fully active in binding GTP, was never found to be fully active in binding aminoacyl-tRNA as judged by high performance liquid chromatography (HPLC) gel filtration and zone-interference gel-electrophoresis. In the presence of kirromycin, however, all these EF-Tu.GTP molecules bind aminoacyl-tRNA, although with a drastically reduced affinity. For the first time, the purification of milligram quantities of ternary complexes of EF-Tu.GTP and aminoacyl-tRNA, free of deacylated tRNA and inactive EF-Tu, has become possible using HPLC gel filtration. We also describe an alternative new method for the isolation of the ternary complexes by means of fractional extraction in the presence of polyethylene glycol. In the latter procedure, the solubility characteristics of the ternary complexes are highly reminiscent to those of free tRNA. Concentrated samples of EF-Tu.GMPPNP.aminoacyl-tRNA complexes show a high stability.     

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.     

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.     

Isolation of Qbeta polymerase complexes containing mutant species of elongation factor Tu

The RNA genome of coliphage Qbeta is replicated by a complex of four proteins, one of them being the translation elongation factor Tu. The role of EF-Tu in this RNA polymerase complex is still unclear, but the obligate presence of translationally functional EF-Tu in the cell hampers the use of conventional mutational analysis. Therefore, we designed a system based on affinity chromatography and could separate two types of complexes by placing an affinity tag on mutated EF-Tu species. Thus, we were able to show a direct link between the vital tRNA binding property of EF-Tu and polymerase activity.     

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).     

NMR study of the phosphate-binding elements of Escherichia coli elongation factor Tu catalytic domain

The phosphoryl-binding elements in the GDP-binding domain of elongation factor Tu were studied by heteronuclear proton observe methods. Five proton resonances were found below 10.5 ppm. Two of these were assigned to the amide groups of Lys 24 and Gly 83. These are conserved residues in each of the consensus sequences. Their uncharacteristic downfield proton shifts are attributed to strong hydrogen bonds to phosphate oxygens as for resonances in N-ras-p21 [Redfield, A. G., & Papastavros, M. Z. (1990) Biochemistry 29, 3509-3514]. The Lys 24 of the EF-Tu G-domain has nearly the same proton and nitrogen shifts as the corresponding Lys 16 in p21. These results suggest that this conserved lysine has a similar structural role in proteins in this class. The tentative Gly 83 resonance has no spectral analogue in p21. A mutant protein with His 84 changed to glycine was fully 15N-labeled and the proton resonance assigned to Gly 83 shifted downfield by 0.3 ppm, thereby supporting the assignment.     

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.     

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.     

T-armless tRNAs and elongated elongation factor Tu

Most tRNAs share a common secondary structure containing a T arm, a D arm, an anticodon arm and an acceptor stem. However, there are some exceptions. Most nematode mitochondrial tRNAs and some animal mitochondrial tRNAs lack the T arm, which is necessary for binding to canonical elongation factor Tu (EF-Tu). The mitochondria of the nematode Caenorhabditis elegans have a unique EF-Tu, named EF-Tu1, whose structure has supplied clues as to how truncated tRNAs can work in translation. EF-Tu1 has a C-terminal extension of about 60 aa that is absent in canonical EF-Tu. Recent data from our laboratory strongly suggests that EF-Tu1 recognizes the D-arm instead of the T arm by a mechanism involving this C-terminal region. Further biochemical analysis of mitochondrial tRNAs and EF-Tu from the distantly related nematode Trichinella spp. and sequence information on nuclear and mitochondrial DNA in arthropods suggest that T-armless tRNAs may have arisen as a result of duplication of the EF-Tu gene. These studies provide valuable insights into the co-evolution of RNA and RNA-binding proteins.     

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.     

Isolation and characterization of Streptomyces aureofaciens protein-synthesis elongation factor Tu in an aggregated state

The ability of EF-Tu to aggregate spontaneously was employed for the purification of homogeneous EF-Tu . GDP from Streptomyces aureofaciens. The formation of filamentous structures in the aggregated EF-Tu was demonstrated in a light microscope. The purified factor, with a specific activity of 19,100 +/- 1,000 units/mg in [3H]GDP exchange, was shown to be active in the translation of poly(U). Aggregated EF-Tu . GDP exhibited almost eight-times lower GDP-exchange capacity at 2 degrees C than at 30 degrees C. This suggests that GDP-binding sites are not freely accessible at lower temperatures in the aggregated factor, in contrast to Escherichia coli polymerized EF-Tu. Turbidimetric assays revealed that the solubilization of diluted aggregated S. aureofaciens EF-Tu is strongly dependent on temperature and causes an increase in the number of accessible GDP-binding sites.     

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).