GE2270A-resistant mutations in elongation factor Tu allow productive aminoacyl-tRNA binding to EF-Tu.GTP.GE2270A complexes

The antibiotic GE2270A prevents stable complex formation between elongation factor Tu (EF-Tu) and aminoacyl-tRNA (aatRNA). In Escherichia coli we characterized two mutant EF-Tu species with either G257S or G275A that lead to high GE2270A resistance in poly(Phe) synthesis, which at least partially explains the high resistance of EF-Tu1 from GE2270A producer Planobispora rosea to its own antibiotic. Both E. coli mutants were unexpectedly found to bind GE2270A nearly as well as wild-type (wt) EF-Tu in their GTP-bound conformations. Both G257S and G275A are in or near the binding site for the 3' end of aatRNA. The G257S mutation causes a 2.5-fold increase in affinity for aatRNA, whereas G275A causes a 40-fold decrease. In the presence of GE2270A, wt EF-Tu shows a drop in aatRNA affinity of at least four orders of magnitude. EF-Tu[G275S] and EF-Tu[G275A] curtail this drop to about two or one order, respectively. It thus appears that the resistance mutations do not prevent GE2270A from binding to EF-Tu.GTP and that the mutant EF-Tus may accommodate GE2270A and aatRNA simultaneously. Interestingly, in their GDP-bound conformations the mutant EF-Tus have much less affinity for GE2270A than wt EF-Tu. The latter is explained by a recent crystal structure of the EF-Tu.GDP.GE2270A complex, which predicts direct steric problems between GE2270A and the mutated G257S or G275A. These mutations may cause a dislocation of GE2270A in complex with GTP-bound EF-Tu, which then no longer prevents aatRNA binding as in the wt situation. Altogether, the data lead to the following novel resistance scenario. Upon arrival of the mutant EF-Tu.GTP.GE2270.aatRNA complex at the ribosomal A-site, the GTPase centre is triggered. The affinities of aatRNA and GE2270A for the GDP-bound EF-Tu are negligible; the former stays at the A-site for subsequent interaction with the peptidyltransferase centre and the latter two dissociate from the ribosome.     

Structural basis of the action of pulvomycin and GE2270 A on elongation factor Tu

Pulvomycin inhibits protein synthesis by preventing the formation of the ternary complex between elongation factor Tu (EF-Tu) x GTP and aa-tRNA. In this work, the crystal structure of Thermus thermophilus EF-Tu x pulvomycin in complex with the GTP analogue guanylyl imino diphosphate (GDPNP) at 1.4 A resolution reveals an antibiotic binding site extending from the domain 1-3 interface to domain 2, overlapping the domain 1-2-3 junction. Pulvomycin binding interferes with the binding of the 3'-aminoacyl group, the acceptor stem, and 5' end of tRNA. Only part of pulvomycin overlaps the binding site of GE2270 A, a domain 2-bound antibiotic of a structure unrelated to pulvomycin, which also hinders aa-tRNA binding. The structure of the T. thermophilus EF-Tu x GDPNP x GE2270 A complex at 1.6 A resolution shows that GE2270 A interferes with the binding of the 3'-aminoacyl group and part of the acceptor stem of aa-tRNA but not with the 5' end. Both compounds, pulvomycin more markedly, hinder the correct positioning of domain 1 over domains 2 and 3 that characterizes the active form of EF-Tu, while they affect the domain 1 switch regions that control the EF-Tu x GDP/GTP transitions in different ways. This work reveals how two antibiotics with different structures and binding modes can employ a similar mechanism of action.     

Nucleotide sequence and codon usage of the elongation factor Tu(EF-Tu) gene from Mycoplasma pneumoniae

The Mycoplasma pneumoniae tuf gene, encoding the elongation factor protein Tu, was cloned and sequenced. The nucleotide sequence of the mycoplasmal gene showed about 60% homology to the sequences of tuf genes of other prokaryotes, yeast mitochondria and Euglena gracilis chloroplasts, and about 75% similarity was found when comparing the deduced amino acid sequences of the various Tu proteins. The relatively low G + C content (40%) of the M. pneumoniae DNA was reflected in a low G + C content (44.6%) of the tuf gene, and in a preferential use of adenine and uracil at the third position of codons, yet codon usage analysis revealed the presence of almost all of the codons of the genetic code in the mycoplasmal gene. Southern blot hybridization of digested DNAs of 11 Mollicutes species with the entire M. pneumoniae tuf gene and with its 5' part suggested the presence of one copy only of this gene in the representative species of the Mollicutes. In this respect, the Mollicutes resemble Gram-positive bacteria and differ from the Gram-negative bacteria, which carry two copies of the tuf gene.     

Immunological heterogeneity of archaebacterial protein synthesis elongation factors Tu (EF-Tu)

Abstract Polyclonal antibodies were raised against the EF-Tu of the archaebacterium Sulfolobus solfataricus and cross-reactivities of EF-Tus of other, phylogenetically disparate archaebacteria were determined using Western blotting and ELISA. The results demonstrate a high degree of heterogeneity of archaebacterial Tu factors with recognition by S. solfataricus EF-Tu antibodies ranging from 48% to 1.5% that observed with the homologous antigen. The immunochemical relatedness between the heterologous and the cognate ( Sulfolobus ) antigens correlates satisfactorily with similarities in 16 S rRNA sequences, there being no recognition of eubacterial and eukaryotic factors by the S. solfataricus EF-Tu antibodies.     

Conformational change of elongation factor Tu (EF-Tu) induced by antibiotic binding. Crystal structure of the complex between EF-Tu.GDP and aurodox

Aurodox is a member of the family of kirromycin antibiotics, which inhibit protein biosynthesis by binding to elongation factor Tu (EF-Tu). We have determined the crystal structure of the 1:1:1 complex of Thermus thermophilus EF-Tu with GDP and aurodox to 2.0-A resolution. During its catalytic cycle, EF-Tu adopts two strikingly different conformations depending on the nucleotide bound: the GDP form and the GTP form. In the present structure, a GTP complex-like conformation of EF-Tu is observed, although GDP is bound to the nucleotide-binding site. This is consistent with previous proposals that aurodox fixes EF-Tu on the ribosome by locking it in its GTP form. Binding of EF-Tu.GDP to aminoacyl-tRNA and mutually exclusive binding of kirromycin and elongation factor Ts to EF-Tu can be explained on the basis of the structure. For many previously observed mutations that provide resistance to kirromycin, it can now be understood how they prevent interaction with the antibiotic. An unexpected feature of the structure is the reorientation of the His-85 side chain toward the nucleotide-binding site. We propose that this residue stabilizes the transition state of GTP hydrolysis, explaining the acceleration of the reaction by kirromycin-type antibiotics.     

Crosslinking of elongation factor Tu to tRNA(Phe) by trans-diamminedichloroplatinum (II). Characterization of two crosslinking sites on EF-Tu

In a preceding paper [(1987) Nucleic Acids Res. 15, 5787-5801], we have used trans-diamminedichloroplatinum (II) to induce reversible RNA-protein crosslinks within the ternary EF-Tu/GTP/Phe-tRNA(Phe) complex and have identified two crosslinking sites on the tRNA. The aim of the present paper is to determine the crosslinking sites on EF-Tu. Two tryptic peptides located in domain I could be identified, a major one (residues 45-74) and a minor one (residues 117-154). The use of Staphylococcus aureus V8 protease led to the isolation of two major peptides (residues 56-68 and 64-68) and one minor peptide (118-124). These results are discussed in the light of the current knowledge of the topography of the EF-Tu/tRNA complex.     

Ef-Ts elongation factor interacts with elongation factor EF-Tu on ribosomes prior to the GTP hydrolysis stage

Methods of high-speed centrifugation and limited proteolysis were used to probe the interaction of EF-Tu with EF-Ts on the ribosome. It is shown that EF-Ts dissociates from EF-Tu only after EF-Tu-mediated GTP hydrolysis, i.e. EF-Ts within the EF-Tu.ribosome complexes in the pre-GTP-hydrolysis state co-sediments with the ribosomes and its rate of proteolysis is distinct from that of free EF-Ts. Moreover, as seen from the difference in sensitivity to trypsin of ribosomal proteins L19 and L27 EF-Ts affects the interaction of EF-Tu with the ribosome.     

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.     

Conformation and reactivity changes induced by N-methylkirromycin (aurodox) in elongation factor Tu

Kirromycin and related antibiotics inhibit protein synthesis in bacteria by acting on elongation factor Tu (EF-Tu). We have studied the effects of N-methylkirromycin (aurodox) on some molecular properties of this protein. The binding of the antibiotic causes a dramatic variation in the protein fluorescence emission spectrum with the appearance of a new maximum at around 340 nm. Addition of aurodox to trypsinized EF-Tu resulted in an emission spectrum similar to that of the denatured intact factor. Fluorescence lifetime analysis performed by a multifrequency phase fluorometer indicated that the fluorescence emission of the factor is heterogeneous with the major component having a lifetime near 4.8 ns in the absence and 6.6 ns in the presence of the antibiotic. These results were interpreted in terms of an antibiotic-induced environmental modification of the unique tryptophan residue of the protein leading to an increase in its quantum yield. However, aurodox did not modify the solvent exposure of this residue, as judged by fluorescence quenching experiments. Moreover, 1-anilino-8-naphthalenesulfonate (ANS) binding studies, as well as analysis of the protein reactivity toward the sulfhydryl group reagent 5,5'-dithiobis(2-nitrobenzoate) (DTNB), showed that, in the presence of aurodox, the behavior of the EF-Tu-GDP complex nears that of EF-Tu.GTP. These results strongly support the hypothesis that aurodox not only confers a "GTP-like" conformation to the EF-Tu.GDP complex but also produces a less stable folding of the protein around the tryptophan residue that may contribute to the multiple functional effects of this antibiotic.     

Antibiotic production improvement in the rare actinomycete Planobispora rosea by selection of mutants resistant to the Aminoglycosides Streptomycin and Gentamycin and to Rifamycin

During a strain improvement program, spontaneous mutants with single or combined resistance to streptomycin (Str^r), gentamycin (Gen^r) or rifamycin (Rif^r) were selected from the industrial strain of Planobispora rosea, which is the producer of thiazolylpeptide GE2270. Among the mutants resistant to each single antibiotic, higher producers occurred more frequently (60%) among Gen^r than in Rif^r (10%) and Str^r (24%) populations. Two Gen^r mutants showed up to 1.5-fold improvement in GE2270 production while single resistant mutants Str^r and Rif^r produced slightly more than the parental strains. The combination of Str^r and Rif^r in the same strain improved GE2270 yield up to 1.7-fold. Finally, a higher GE2270 producing strain (1.8-fold improvement with respect to the parental strain) was selected among those mutants with triple resistance to streptomycin, rifamycin and gentamycin. A hierarchical increase in aerial mycelium and spore formation was observed which paralleled GE2270 production improvement.     

Interaction of apicoplast-encoded elongation factor (EF) EF-Tu with nuclear-encoded EF-Ts mediates translation in the Plasmodiumfalciparum plastid

Protein translation in the plastid (apicoplast) of Plasmodium spp. is of immense interest as a target for potential anti-malarial drugs. However, the molecular data on apicoplast translation needed for optimisation and development of novel inhibitors is lacking. We report characterisation of two key translation elongation factors in Plasmodium falciparum, apicoplast-encoded elongation factor PfEF-Tu and nuclear-encoded PfEF-Ts. Recombinant PfEF-Tu hydrolysed GTP and interacted with its presumed nuclear-encoded partner PfEF-Ts. The EF-Tu inhibitor kirromycin affected PfEF-Tu activity in vitro, indicating that apicoplast EF-Tu is indeed the target of this drug. The predicted PfEF-Ts leader sequence targeted GFP to the apicoplast, confirming that PfEF-Ts functions in this organelle. Recombinant PfEF-Ts mediated nucleotide exchange on PfEF-Tu and homology modeling of the PfEF-Tu:PfEF-Ts complex revealed PfEF-Ts-induced structural alterations that would expedite GDP release from PfEF-Tu. Our results establish functional interaction between two apicoplast translation factors encoded by genes residing in different cellular compartments and highlight the significance of their sequence/structural differences from bacterial elongation factors in relation to inhibitor activity. These data provide an experimental system to study the effects of novel inhibitors targeting PfEF-Tu and PfEF-Tu.PfEF-Ts interaction. Our finding that apicoplast EF-Tu possesses chaperone-related disulphide reductase activity also provides a rationale for retention of the tufA gene on the plastid genome.     

Mapping the effector region in Thermus thermophilus elongation factor Tu

Native elongation factor Tu from Thermus thermophilus is initially attacked by various endoproteases in a region spanning amino acid residues 40-70. By comparing the hydrolysis rates of nucleotide-free and GDP-bound EF-Tu, only a small difference was observed for the tryptic cleavage at Arg-59. Protease V-8 attacks Glu-55 only in a GDP/GTP form, whereas this enzyme exclusively hydrolyze Asn-64 in nucleotide-free EF-Tu, even when the protein had been previously cleaved at Arg-59. Binding of GDP leads to a 42-fold decreased rate of hydrolysis by the Lys-C protease at Lys-52. It also reduces the accessibility of Lys-275 to trypsin, reflecting a "long-range" effect from nucleotide binding domain I to domain II. Only slight differences were observed in the rate of hydrolysis at all positions in the GDP- versus the GTP-bound form. The intrinsic GTPase activity was slightly reduced in trypsin-treated EF-Tu, significantly impaired in EF-Tu cleaved at Lys-52, and completely abolished in EF-Tu cleaved at Asn-64. No ribosome-induced GTPase activity was observed for protease-cleaved EF-Tu's. Treatment of these proteins with periodate-oxidized GDP or GTP followed by cyanoborohydride led to covalent modification of the new N-terminus located exclusively within region 52-60. The highest reactivity was shown by the N-terminus of Glu-56. Additionally, lysine residues in the native protein sensitive to affinity labeling [Peter, M.E., Wittmann-Liebold, B., & Sprinzl, M. (1988) Biochemistry 27, 9132-9139] lost their reactivity upon cleavage of EF-Tu in region 52-60, suggesting an altered structure of the cleaved protein.(ABSTRACT TRUNCATED AT 250 WORDS)     

An "elongated" translation elongation factor Tu for truncated tRNAs in nematode mitochondria

We have found the gene for a translation elongation factor Tu (EF-Tu) homologue in the genome of the nematode Caenorhabditis elegans. Because the corresponding protein was detected immunologically in a nematode mitochondrial (mt) extract, it could be regarded as a nematode mt EF-Tu. The protein possesses an extension of about 57 amino acids (we call this domain 3') at the C terminus, which is not found in any other known EF-Tu. Because most nematode mt tRNAs lack a T stem, domain 3' may be related to this feature. The nematode EF-Tu bound to nematode T stem-lacking tRNA, but bacterial EF-Tu was unable to do so. A series of domain exchange experiments strongly suggested that domains 3 and 3' are essential for binding to T stem-lacking tRNAs. This finding may constitute a novel example of the co-evolution of a structurally simplified RNA and the cognate RNA-binding protein, the latter having apparently acquired an additional domain to compensate for the lack of a binding site(s) on the RNA.     

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.