Domain III of elongation factor G from Thermus thermophilus is essential for induction of GTP hydrolysis on the ribosome

Two elongation factors (EF) EF-Tu and EF-G participate in the elongation phase during protein biosynthesis on the ribosome. Their functional cycles depend on GTP binding and its hydrolysis. The EF-Tu complexed with GTP and aminoacyl-tRNA delivers tRNA to the ribosome, whereas EF-G stimulates translocation, a process in which tRNA and mRNA movements occur in the ribosome. In the present paper we report that: (a) intrinsic GTPase activity of EF-G is influenced by excision of its domain III; (b) the EF-G lacking domain III has a 10(3)-fold decreased GTPase activity on the ribosome, whereas its affinity for GTP is slightly decreased; and (c) the truncated EF-G does not stimulate translocation despite the physical presence of domain IV, which is also very important for translocation. By contrast, the interactions of the truncated factor with GDP and fusidic acid-dependent binding of EF-G.GDP complex to the ribosome are not influenced. These findings indicate an essential contribution of domain III to activation of GTP hydrolysis. These results also suggest conformational changes of the EF-G molecule in the course of its interaction with the ribosome that might be induced by GTP binding and hydrolysis.     

Structure and Functions of the Prokaryotic Elongation Factor G

Structural and functional data on elongation factor G (EF-G) are reviewed with regard to nucleotide exchange, GTP hydrolysis, mechanism of action of fusidic acid, and functional roles of the EF-G structural domains in translocation. Biochemical data are correlated with structural dynamics of the EF-G molecule on interaction with various ligands. Data on EF-Tu are also considered, as EF-G and EF-Tu share certain structural and functional features.     

Structure and functions of the prokaryotic elongation factor G

Structural and functional data on elongation factor G (EF-G) are reviewed with regard to nucleotide exchange, GTP hydrolysis, mechanism of action of fusidic acid, and functional roles of the EF-G structural domains in translocation. Biochemical data are correlated with structural dynamics of the EF-G molecule on interaction with various ligands. Data on EF-Tu are also considered, as EF-G and EF-Tu share certain structural and functional features.     

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.     

The eubacterial protein synthesis inhibitor pulvomycin interacts with archaeal elongation factor 1α from Sulfolobus solfataricus

The effect of pulvomycin on the biochemical and fluorescence spectroscopic properties of the archaeal elongation factor 1α from Sulfolobus solfataricus (SsEF-1α), the functional analog of eubacterial EF-Tu, was investigated. The antibiotic was able to reduce in vitro the rate of protein synthesis however, the concentration of pulvomycin leading to 50% inhibition (173 μM) was two order of magnitude higher but one order lower than that required in eubacteria and eukarya, respectively. The effect of the antibiotic on the partial reactions catalysed by SsEF-1α indicated that pulvomycin was able to decrease the affinity of the elongation factor toward aa-tRNA only in the presence of GTP, to an extent similar to that measured in the presence of GDP. Moreover, the antibiotic produced an increase of the intrinsic GTPase catalysed by SsEF-1α, but not that of its engineered forms. Finally, pulvomycin induced a variation in fluorescence spectrum of the aromatic region of the elongation factor and its truncated forms. These spectroscopic results suggested that a conformational change of the elongation factor takes place upon interaction with the antibiotic. This finding was confirmed by the protection against chemical denaturation of SsEF-1α, observed in the presence of pulvomycin. However, a stabilising effect of the antibiotic directly on the protein in the complex could takes place.     

Initiation factor IF2, thiostrepton and micrococcin prevent the binding of elongation factor G to the Escherichia coli ribosome

The bacterial translational GTPases (initiation factor IF2, elongation factors EF-G and EF-Tu and release factor RF3) are involved in all stages of translation, and evidence indicates that they bind to overlapping sites on the ribosome, whereupon GTP hydrolysis is triggered. We provide evidence for a common ribosomal binding site for EF-G and IF2. IF2 prevents the binding of EF-G to the ribosome, as shown by Western blot analysis and fusidic acid-stabilized EF-G.GDP.ribosome complex formation. Additionally, IF2 inhibits EF-G-dependent GTP hydrolysis on 70 S ribosomes. The antibiotics thiostrepton and micrococcin, which bind to part of the EF-G binding site and interfere with the function of the factor, also affect the function of IF2. While thiostrepton is a strong inhibitor of EF-G-dependent GTP hydrolysis, GTP hydrolysis by IF2 is stimulated by the drug. Micrococcin stimulates GTP hydrolysis by both factors. We show directly that these drugs act by destabilizing the interaction of EF-G with the ribosome, and provide evidence that they have similar effects on IF2.     

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.     

The highly conserved LepA is a ribosomal elongation factor that back-translocates the ribosome

The ribosomal elongation cycle describes a series of reactions prolonging the nascent polypeptide chain by one amino acid and driven by two universal elongation factors termed EF-Tu and EF-G in bacteria. Here we demonstrate that the extremely conserved LepA protein, present in all bacteria and mitochondria, is a third elongation factor required for accurate and efficient protein synthesis. LepA has the unique function of back-translocating posttranslocational ribosomes, and the results suggest that it recognizes ribosomes after a defective translocation reaction and induces a back-translocation, thus giving EF-G a second chance to translocate the tRNAs correctly. We suggest renaming LepA as elongation factor 4 (EF4).     

Archaebacterial elongation factor Tu insensitive to pulvomycin and kirromycin

A spermine-dependent, polyphenylalanine-synthesizing cell-free system having an optimum activity at 75-85 degrees C, has been developed from the extremely thermoacidophilic archaebacterium Caldariella acidophila. The C. acidophila system is totally insensitive to the EF-Tu targeted antibiotics pulvomycin (at 40 degrees C) and kirromycin (at 47-72 degrees C) contrary to control systems derived from both mesophilic (Escherichia coli) and thermoacidophilic (Bacillus acidocaldarius) eubacteria. The archaebacterial EF-Tu-equivalent factor is also immunologically unrelated to eubacterial EF-Tu and does not cross react with antibodies against Escherichia coli EF-Tu. The pulvomycin and kirromycin reactions thus provide new phyletic markers for archaebacterial ancestry.     

Bovine mitochondrial ribosomes. Elongation factor specificity

The activity of bovine mitochondrial ribosomes with elongation factors from a variety of sources including the mitochondria of lower eukaryotes, chloroplasts, Gram-negative bacteria, Gram-positive bacteria, and the eukaryotic cell cytoplasm has been investigated. Bovine mitochondrial ribosomes are active with homologous mitochondrial elongation factor (EF)-G but display no activity with the mitochondrial or chloroplast translocases from the lower eukaryote Euglena gracilis, with Escherichia coli or Bacillus subtilis EF-G or with cytoplasmic EF-2. In contrast to the results obtained with the translocases, E. coli EF-Tu, B. subtilis EF-Tu, and Euglena chloroplast EF-Tu all function to a significant extent on the mitochondrial ribosomes. Cytoplasmic EF-1 has barely detectable activity on the animal mitochondrial ribosomes. The polymerization of phenylalanine by these ribosomes is dependent on poly(U), displays a rather broad Mg2+ optimum around 12 mM, and proceeds most rapidly at low monovalent ion concentrations.     

GTPases mechanisms and functions of translation factors on the ribosome

The elongation factors (EF) Tu and G and initiation factor 2 (IF2) from bacteria are multidomain GTPases with essential functions in the elongation and initiation phases of translation. They bind to the same site on the ribosome where their low intrinsic GTPase activities are strongly stimulated. The factors differ fundamentally from each other, and from the majority of GTPases, in the mechanisms of GTPase control, the timing of Pi release, and the functional role of GTP hydrolysis. EF-Tu x GTP forms a ternary complex with aminoacyl-tRNA, which binds to the ribosome. Only when a matching codon is recognized, the GTPase of EF-Tu is stimulated, rapid GTP hydrolysis and Pi release take place, EF-Tu rearranges to the GDP form, and aminoacyl-tRNA is released into the peptidyltransferase center. In contrast, EF-G hydrolyzes GTP immediately upon binding to the ribosome, stimulated by ribosomal protein L7/12. Subsequent translocation is driven by the slow dissociation of Pi, suggesting a mechano-chemical function of EF-G. Accordingly, different conformations of EF-G on the ribosome are revealed by cryo-electron microscopy. GTP hydrolysis by IF2 is triggered upon formation of the 70S initiation complex, and the dissociation of Pi and/or IF2 follows a rearrangement of the ribosome into the elongation-competent state.     

Genetic and biochemical characterization of kirromycin resistance mutations in Bacillus subtilis.

Spontaneous mutations causing resistance to the EF-Tu-specific antibiotic kirromycin have been isolated and mapped in Bacillus subtilis. Three-factor transductional and transformational crosses have placed the kir locus proximal to ery-1 and distal to strA (rpsL) and several mutations affecting elongation factors EF-G and EF-Tu, in the order: cysA strA [fus-1/ts-6(EF-G)] [ts-5(EF-Tu)] kir ery-1 spcA. Purified EF-Tu from mutant strains is more resistant to kirromycin as measured by in vitro protein synthesis and also shows a more acidic isoelectric point than wild-type EF-Tu. This indicates that the kir locus is the genetic determinant (tuf) for EF-Tu and that there is a single active gene for this enzyme in B. subtilis.     

Mechanism of natural resistance to kirromycin-type antibiotics in actinomycetes

Abstract The sensitivity of intact cells and subcellular fractions of actinomycetes to kirromycin and pulvomycin was examined. These antibiotics block bacterial protein synthesis by acting on elongation factor Tu (EF-Tu). Two types of natural resistance were encountered in actinomycetes. Some strains were resistant to kirromycin and pulvomycin by virtue of inefficient cellular uptake of these drugs. In 3 strains, kirromycin resistance was attributable to a drug-insensitive EF-Tu. These 3 organisms produce kirromycin-type antibiotics: Streptomyces cinnamomeus, Streptomyces lactamdurans and Streptoverticillium mobaraense synthesize kirrothricin, efrotomycin and pulvomycin, respectively. In S. cinnamomeus and S. lactamdurans resistance to their own antibiotic is due to possession of a nonresponding EF-Tu factor, whereas pulvomycin resistance in Sv. mobaraense is more likely derived from the permeability properties of the cell envelope.     

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.     

Elongation factor-dependent affinity labeling of Escherichia coli ribosomes

Elongation factor-dependent affinity labeling of Escherichia coli ribosomes was obtained using a functional analogue of aminoacyl-tRNA. Since elongation factor Tu (EF-Tu) screens both the modified aminoacyl-tRNAs and the ribosomal complexes for active particles, only functional macromolecular complexes are examined. This approach also provides an unequivocal identification of the transfer RNA binding site from which affinity labeling occurs. N^ε-bromoacetyl-Lys-tRNA was prepared by covalently attaching an electrophilic group to the side-chain of the amino acid. This chemical modification did not interfere with function, since the ?BrAcLys-tRNA participated successfully in EF-Tu and poly(rA)-dependent binding to ribosomes, peptide bond formation, and elongation factor G (EF-G)-mediated translocation. Affinity labeling of ribosomal RNA was observed only in those incubations which contained both EF-Tu and EF-G. The crosslinking of ?BrAcLys-tRNA to 23 S rRNA was found even if fusidic acid was added to the incubation before EF-G. The dependence of the covalent reaction on EF-G demonstrates, unambiguously, that a reactive residue of 23 S rRNA is located adjacent to the 3′ end of the functionally defined P site. Similarly, the affinity labeling of proteins L13/14/15, L2, L32/33, and L24 required EF-G-dependent translocation of ?BrAcLys-tRNA into the P site. Protein L27 was alkylated following the EF-Tu-dependent binding of ?BrAcLys-tRNA to the ribosome, and the extent of affinity labeling was stimulated by the addition of EF-G to the incubation. Double-label dipeptide experiments confirmed that affinity labeling occurred from functional tRNA binding sites by demonstrating that the same ?BrAcLys-tRNA which reacted covalently with 23 S rRNA or a ribosomal protein could also participate in peptide bond formation. Finally, the ribosome affinity labeling obtained with ?BrAcLys-tRNA · EF-Tu · guanylylimidodiphosphate differed little from that obtained with ?BrAcLys-tRNA · EF-Tu · GTP. This work constitutes the first direct examination of the aminoacyl ends of the EF-Tu-dependent conformational states of the ribosomal complex, and demonstrates the potential value of functional Lys-tRNA analogues with different probes attached to the lysine side-chain.     

Kirromycin-resistant elongation factor Tu from pulvomycin-producing wild-type of streptoverticillium mobaraense

Elongation factor Tu (EF-Tu) ofStreptoverticillium mobaraense, which produces pulvomycin, has been prepared to 90% purity. The purified protein differs significantly from the analogous protein found inEscherichia coli in molecular weight and antibiotic sensitivity. EF-Tu migrates in sodium dodecyl sulfate gel electrophoresis as a 46,000-dalton species. The protein is sensitive to pulvomycin, but highly resistant to kirromycin. EF-Tu fromStv. mobaraense exists in multiple forms as monomer and polymers. By contrast to the monomer, the polymers of EF-Tu are completely resistant to pulvomycin.     

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.     

The antibiotics kirromycin and pulvomycin bind to different sites on the elongation factor Tu from Escherichia coli

Pulvomycin and kirromycin, two antibiotics which inhibit protein biosynthesis in Escherichia coli by complex formation with the elongation factor Tu (EF-Tu), bind to different sites on the protein. While only one molecule of kirromycin can be bound to one molecule of EF-Tu, more than one molecule of pulvomycin interacts with a molecule of EF-Tu. This has been deduced from experiments in which the aminoacyl-tRNA binding and the GTPase activity of EF-Tu were measured in the presence of varying amounts of both antibiotics. These experiments are interpreted to mean that pulvomycin but not kirromycin can replace the other antibiotic in its respective site. Our conclusions are supported by circular dichroism spectroscopy.     

In vivo selection of conditional-lethal mutations in the gene encoding elongation factor G of Escherichia coli.

The ribosome translocation step that occurs during protein synthesis is a highly conserved, essential activity of all cells. The precise movement of one codon that occurs following peptide bond formation is regulated by elongation factor G (EF-G) in eubacteria or elongation factor 2 (EF-2) in eukaryotes. To begin to understand molecular interactions that regulate this process, a genetic selection was developed with the aim of obtaining conditional-lethal alleles of the gene (fusA) that encodes EF-G in Escherichia coli. The genetic selection depends on the observation that resistant strains arose spontaneously in the presence of sublethal concentrations of the antibiotic kanamycin. Replica plating was performed to obtain mutant isolates from this collection that were restrictive for growth at 42 degrees C. Two tightly temperature-sensitive strains were characterized in detail and shown to harbor single-site missense mutations within fusA. The fusA100 mutant encoded a glycine-to-aspartic acid change at codon 502. The fusA101 allele encoded a glutamine-to-proline alteration at position 495. Induction kinetics of beta-galactosidase activity suggested that both mutations resulted in slower elongation rates in vivo. These missense mutations were very near a small group of conserved amino acid residues (positions 483 to 493) that occur in EF-G and EF-2 but not EF-Tu. It is concluded that these sequences encode a specific domain that is essential for efficient translocase function.