Recognition of the universally conserved 3'-CCA end of tRNA by elongation factor EF-Tu.

Escherichia coli tRNA(Val) with pyrimidine substitutions for the universally conserved 3'-terminal adenine can be readily aminoacylated. It cannot, however, transfer valine into polypeptides. Conversely, despite being a poor substrate for valyl-tRNA synthetase, tRNA(Val) with a 3'-terminal guanine is active in in vitro polypeptide synthesis. To better understand the function of the 3'-CCA sequence of tRNA in protein synthesis, the effects of systematically varying all three bases on formation of the Val-tRNA(Val):EF-Tu:GTP ternary complex were investigated. Substitutions at C74 and C75 have no significant effect, but replacing A76 with pyrimidines decreases the affinity of valyl-tRNA(Val) for EF-Tu:GTP, thus explaining the inability of these tRNA(Val) variants to function in polypeptide synthesis. Valyl-tRNA(Val) terminating in 3'-guanine is readily recognized by EF-TU:GTP. Dissociation constants of the EF-Tu:GTP ternary complexes with valine tRNAs having nucleotide substitutions at the 3' end increase in the order adenine < guanine < uracil; EF-Tu has very little affinity for tRNA terminating in 3' cytosine. Similar observations were made in studies of the interaction of 3' end mutants of E. coli tRNA(Ala) and tRNA(Phe) with EF-Tu:GTP. These results indicate that EF-Tu:GTP preferentially recognizes purines and discriminates against pyrimidines, especially cytosine, at the 3' end of aminoacyl-tRNAs.     

Interaction of tRNA with 23S rRNA in the ribosomal A, P, and E sites

Three sets of conserved nucleotides in 23 rRNA are protected from chemical probes by binding of tRNA to the ribosomal A, P, and E sites, respectively. They are located almost exclusively in domain V, primarily in or adjacent to the loop identified with the peptidyl transferase function. Some of these sites are also protected by antibiotics such as chloramphenicol, which could explain how these drugs interfere with protein synthesis. Certain tRNA-dependent protections are abolished when the 3'-terminal A or CA or 2',3'-linked acyl group is removed, providing direct evidence for the interaction of the conserved CCA terminus of tRNA with 23S rRNA. When the EF-Tu.GTP.aminoacyl-tRNA ternary complex is bound to the ribosome, no tRNA-dependent A site protections are detected in 23S rRNA until EF-Tu is released. Thus, EF-Tu prevents interaction of the 3' terminus of the incoming aminoacyl-tRNA with the peptidyl transferase region of the ribosome during anticodon selection, thereby permitting translational proofreading.     

Role of the 5'-terminal phosphate of tRNA for its function during protein biosynthesis elongation cycle.

The 5'-terminal phosphate of tRNAPhe from yeast was removed using tRNAPhe lacking its 3'-terminal adenosine. After regeneration of the C-C-A terminus this tRNA was investigated in following reactions: aminoacylation, spontaneous hydrolysis of the amino acid from aminoacyl-tRNA, aminoacyl-tRNA.EF-Tu.GTP ternary complex formation and poly(U)-dependent synthesis of poly(Phe). The absence of the 5'-terminal phosphate of Phe-tRNAPhe does not influence the rate of hydrolysis of the amino acid or the ability of this rRNA to participate in complex formation with EF-Tu.GTP. The translation of the polyuridylic acid is slightly inhibited whereas the rate and extent of the enzymatic aminoacylation is not affected.     

Specificity of elongation factor EF-TU for hydrophobic peptides

The elongation factor EF-Tu carries aminoacyl-tRNAs to the A-site of the ribosome during the elongation process of protein biosynthesis. We, and others, have recently reported that the Escherichia coli EF-Tu interacts with unfolded and denatured proteins and behaves like a chaperone in protein folding and protection against protein thermal denaturation. In this study, we have identified EF-Tu binding sites in protein substrates by screening cellulose-bound peptides scanning the sequences of several proteins. The binding motifs recognized by EF-Tu in protein substrates are also recognized by the chaperone DnaK and mainly consist of hydrophobic clusters. EF-Tu interacts as efficiently as DnaK with the membrane spanning sequence of the membrane protein phospholemman and with the signal sequence of alkaline phosphatase. It interacts less efficiently with several other hydrophobic clusters of lysozyme and alkaline phosphatase, which are also DnaK substrates and fails to bind to several DnaK binding sites. Our results suggest that EF-Tu, like DnaK, interacts albeit more weakly with the hydrophobic regions of substrate protein and are consistent with the hypothesis that it possesses chaperone properties.     

The elongation factor Tu binds aminoacyl-tRNA in the presence of GDP

Escherichia coli elongation factor (EF-Tu) binds aminoacyl-tRNAs (aa-tRNA) not only in the presence of GTP but also in the presence of GDP. Complex formation leads to a protection of the aa-tRNA against nonenzymatic deacylation and digestion by pancreatic ribonuclease, as well as to a protection of EF-Tu against proteolysis by trypsin. The equilibrium constant for the binding of Phe-tRNAPheyeast for example to EF-Tu.GDP has been determined to be 0.7 X 10(5) M-1 which is 2 orders of magnitude lower than the equilibrium constant for Phe-tRNAPheyeast binding to EF-Tu.GTP. In the presence of kirromycin, aminoacyl-tRNA binding to EF-Tu.GDP is not affected as much: Phe-tRNAPheyeast is bound with an equilibrium constant of 3 X 10(5) M-1. While there is also a measurable interaction between EF-Tu.GTP and tRNA, such an interaction cannot be detected with EF-Tu.GDP and tRNA, not even at millimolar concentrations. A so far undetected complex formation between aminoacyl-tRNA and EF-Tu.GTP in the presence of pulvomycin, however, could be detected. The results are discussed in terms of the structural requirements of ternary complex formation and in the light of proofreading schemes involving A-site binding on the E. coli ribosome.     

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)     

The elongation factor Tu from Escherichia coli, aminoacyl-tRNA, and guanosine tetraphosphate form a ternary complex which is bound by programmed ribosomes

The interaction of the Escherichia coli elongation factor Tu guanosine tetraphosphate complex (EF-Tu ppGpp) with aminoacyl-tRNAs(aa-tRNA) was reinvestigated by gel filtration and hydrolysis protection experiments. These experiments show that EF-Tu X ppGpp like EF-Tu X GDP (Pingoud, A., Block, W., Wittinghofer, A., Wolf, H. & Fischer, E. (1982) J. Biol. Chem. 257, 11261-11267) forms a fairly stable complex with Phe-tRNAPhe, KAss being 0.6 X 10(5) M-1 at 25 degrees C. The binding of the EF-Tu X ppGpp X aa-tRNA complex to programmed ribosomes was investigated by a centrifugation technique. It is shown that this complex is bound codon-specific with KAss = 3 X 10(7) M-1 at 0 degrees C and that it stimulates peptidyl transfer. A numerical estimation of the intracellular concentration of EF-Tu X GTP X aa-tRNA and EF-Tu X ppGpp X aa-tRNA during normal growth and under the stringent response indicates that ppGpp accumulation does affect the EF-Tu X GTP X aa-tRNA concentration but does not lead to major depletion of this pool. Furthermore, due to the higher affinity of EF-Tu X GTP to aa-tRNA and of the ternary complex EF-Tu X GTP X aa-tRNA to the ribosome, EF-Tu X ppGpp X aa-tRNA binding to the ribosome is not significant. According to our measurements and calculations, therefore, a direct participation of EF-Tu in slowing down the rate of protein biosynthesis and improving its accuracy during amino acid starvation is not obvious.     

A unique tRNA recognition mechanism of Caenorhabditis elegans mitochondrial EF-Tu2

Nematode mitochondria expresses two types of extremely truncated tRNAs that are specifically recognized by two distinct elongation factor Tu (EF-Tu) species named EF-Tu1 and EF-Tu2. This is unlike the canonical EF-Tu molecule that participates in the standard protein biosynthesis systems, which basically recognizes all elongator tRNAs. EF-Tu2 specifically recognizes Ser-tRNA^Ser that lacks a D arm but has a short T arm. Our previous study led us to speculate the lack of the D arm may be essential for the tRNA recognition of EF-Tu2. However, here, we showed that the EF-Tu2 can bind to D arm-bearing Ser-tRNAs, in which the D–T arm interaction was weakened by the mutations. The ethylnitrosourea-modification interference assay showed that EF-Tu2 is unique, in that it interacts with the phosphate groups on the T stem on the side that is opposite to where canonical EF-Tu binds. The hydrolysis protection assay using several EF-Tu2 mutants then strongly suggests that seven C-terminal amino acid residues of EF-Tu2 are essential for its aminoacyl-tRNA-binding activity. Our results indicate that the formation of the nematode mitochondrial (mt) EF-Tu2/GTP/aminoacyl-tRNA ternary complex is probably supported by a unique interaction between the C-terminal extension of EF-Tu2 and the tRNA.     

IgG and IgG2 antibodies from cattle naturally infected with Anaplasma marginale recognize the recombinant vaccine candidate antigens VirB9, VirB10, and elongation factor-Tu

Anaplasma marginale is an important vector-borne rickettsia of ruminants in tropical and subtropical regions of the world. Immunization with purified outer membranes of this organism induces protection against acute anaplasmosis. Previous studies, with proteomic and genomic approach identified 21 proteins within the outer membrane immunogen in addition to previously characterized major surface protein1a-5 (MSP1a-5). Among the newly described proteins were VirB9, VirB10, and elongation factor-Tu (EF-Tu). VirB9, VirB10 are considered part of the type IV secretion system (TFSS), which mediates secretion or cell-to-cell transfer of macromolecules, proteins, or DNA-protein complexes in Gram-negative bacteria. EF-Tu can be located in the bacterial surface, mediating bacterial attachment to host cells, or in the bacterial cytoplasm for protein synthesis. However, the roles of VirB9, VirB10, and TFSS in A. marginale have not been defined. VirB9, VirB10, and EF-Tu have not been explored as vaccine antigens. In this study, we demonstrate that sera of cattle infected with A. marginale, with homologous or heterologous isolates recognize recombinant VirB9, VirB10, and EF-Tu. IgG2 from naturally infected cattle also reacts with these proteins. Recognition of epitopes by total IgG and by IgG2 from infected cattle with A. marginale support the inclusion of these proteins in recombinant vaccines against this rickettsia.     

In vitro methylation of the elongation factor EF-Tu from Escherichia coli

The in vitro methylation of the elongation factor EF-Tu from Escherichia coli was investigated. The methylation of newly synthesized EF-Tu was obtained using lambda rifd 18 DNA as template and S-adenosyl [methyl-3H]methionine as methyl donor. About 3 mol methyl residues were incorporated for every 10 mol EF-Tu synthesized. Analysis of the nature of the methyl-containing residues by protein hydrolysis followed by paper chromatography showed that both mono- and dimethyllysine were present. The methylation of EF-Tu was also studied separately from its synthesis by using cell-free systems with artificially undermethylated components.     

Base 2661 in Escherichia coli 23S rRNA influences the binding of elongation factor Tu during protein synthesis in vivo.

The binding of the EF-Tu.GTP.aminoacyl-tRNA ternary complex (EF, elongation factor) to the ribosome is known to be strengthened by a 2661G-to-C mutation in 23S ribosomal RNA, whereas the binding to normal ribosomes is weakened if the factor is in an appropriate mutant form (Aa). In this report we describe the mutual effects by the 2661C alteration in 23S rRNA and EF-Tu(Aa) on bacterial viability and translation efficiency in strains with normal or mutationally altered ribosomes. The rrnB(2661C) allele on a multicopy plasmid was introduced by transformation into Escherichia coli K-12 strains, harbouring either the wild-type or the mutant gene (tufA) for EF-Tu as well as normal or mutant ribosomal protein S12 (rpsL). Together with wild-type EF-Tu, the 2661C mutant ribosomes decreased the translation elongation rate in a rpsL+ strain or a non-restrictive rpsL224 strain. This reduction was not seen in strains which harbored EF-Tu(Aa) instead of EF-Tu(As) (As, wild-type form). Nonsense codon suppression by tyrT(Su3) suppressor tRNA was reduced by 2661C in a rpsL224 strain in the presence of EF-Tu(As) but not in the presence of EF-Tu(Aa). The lethal effect obtained by the combination of 2661C and a restrictive ribosomal protein S12 mutation (rpsL282) disappeared if EF-Tu(As) was replaced by EF-Tu(Aa) in the strain. In such a viable strain, 2661C had no effect on either the translation elongation rate or nonsense codon suppression. Our data suggest that the G base at position 2661 in 23S rRNA is important for binding of EF-Tu during protein synthesis in vivo. The interaction between this base and EF-Tu is strongly influenced by the structure of ribosomal protein S12.     

Control of the tRNA-tufB operon in Escherichia coli. 3. Feedback inhibition of tufB expression by an EF-Tu with a deletion in the guanine-nucleotide-binding domain

The expression of tufB, one of the two EF-Tu-encoding genes in Escherichia coli, is under autogenous control. Feedback inhibition of tufB expression by plasmid-borne EF-Tu has been used to answer the question of whether or not the integrity of the guanine-nucleotide-binding domain of EF-Tu is required for the autoregulatory role of the factor protein. We show that a large deletion of tufB, causing the elimination of an 81-amino-acid segment from the plasmid-borne EF-Tu, does not abolish tufB repression. We conclude that the autoregulation of the cellular EF-Tu level is not dependent on an intact guanine-nucleotide-binding domain and does not require binding of GTP to EF-Tu. The repressor activity of the deletion derivative of EF-Tu can be measured despite a rapid disappearance of the (altered) mutant protein from the soluble cytoplasmic fraction of the cell. Degradation and assembly in larger complexes are responsible for this disappearance.     

Effects of the antibiotic pulvomycin on the elongation factor Tu-dependent reactions. Comparison with other antibiotics

The antibiotic pulvomycin is an inhibitor of protein synthesis that prevents the formation of the ternary complex between elongation factor (EF-) Tu.GTP and aminoacyl-tRNA. In this report, novel aspects of its action on EF-Tu are described. Pulvomycin markedly affects the equilibrium and kinetics of the EF-Tu-nucleotide interaction, particularly of the EF-Tu.GTP complex. The binding affinity of EF-Tu for GTP is increased 1000 times, mainly as the consequence of a dramatic decrease in the dissociation rate of this complex. In contrast, the affinity for GDP is decreased 10-fold due to a marked increase in the dissociation rate of EF-Tu.GDP (25-fold) that mimics the action of EF-Ts, the GDP/GTP exchange factor of EF-Tu. The effects of pulvomycin and EF-Ts can coexist and are simply additive, supporting the conclusion that these two ligands interact with different sites of EF-Tu. This is further confirmed on native PAGE by the ability of EF-Tu to bind the EF-Ts and the antibiotic simultaneously. Pulvomycin enhances the intrinsic EF-Tu GTPase activity, like kirromycin, though to a much more modest extent. As with kirromycin, this stimulation depends on the concentration and nature of the monovalent cations, Li(+) being the most effective one, followed by Na(+), K(+), and NH(4)(+). In the presence of pulvomycin (in contrast to kirromycin), aa-tRNA and/or ribosomes do not enhance the GTPase activity of EF-Tu. The property of pulvomycin to modify selectively the conformation(s) of EF-Tu is also supported by its effect on heat- and urea-dependent denaturation, and tryptic digestion of the protein. Specific differences and similarities between the action of pulvomycin and the other EF-Tu-specific antibiotics are described and discussed.     

Functional-structural analysis of threonine 25, a residue coordinating the nucleotide-bound magnesium in elongation factor Tu

Elongation factor (EF) Tu Thr-25 is a key residue binding the essential magnesium complexed to nucleotide. We have characterized mutations at this position to the related Ser and to Ala, which abolishes the bond to Mg2+, and a double mutation, H22Y/T25S. Nucleotide interaction was moderately destabilized in EF-Tu(T25S) but strongly in EF-Tu(T25A) and EF-Tu(H22Y/T25S). Binding Phe-tRNAPhe to poly(U).ribosome needed a higher magnesium concentration for the latter two mutants but was comparable at 10 mM MgCl2. Whereas EF-Tu(T25S) synthesized poly(Phe), as effectively as wild type, the rate was reduced to 50% for EF-Tu(H22Y/T25S) and was, surprisingly, still 10% for EF-Tu(T25A). In contrast, protection of Phe-tRNAPhe against spontaneous hydrolysis by the latter two mutants was very low. The intrinsic GTPase in EF-Tu(H22Y/T25S) and (T25A) was reduced, and the different responses to ribosomes and kirromycin suggest that stimulation by these two agents follows different mechanisms. Of the mutants, only EF-Tu(T25A) forms a more stable complex with EF-Ts than wild type. This implies that stabilization of the EF-Tu.EF-Ts complex is related to the inability to bind Mg2+, rather than to a decreased nucleotide affinity. These results are discussed in the light of the three-dimensional structure. They emphasize the importance of the Thr-25-Mg2+ bond, although its absence is compatible with protein synthesis and thus with an active overall conformation of EF-Tu.     

Protein synthesis elongation factor Tu present in spores of<Emphasis Type="Italic">Streptomyces coelicolor</Emphasis> can be phosphorylated<Emphasis Type="Italic">in vitro</Emphasis> by the spore protein kinase

In vitro phosphorylation of EF-Tu was shown in cell-free extract from dormant spores of Streptomyces coelicolor by a protein kinase present in spores. EF-Tu phosphorylation was observed on both intrinsic S. coelicolor factor and externally added purified EF-Tu from S. aureofaciens, on two isoforms. Putative serine and threonine residues as potential phosphorylation targets were determined in primary sequence and demonstrated on 3D structure model of EF-Tu.