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.     

Eukaryotic elongation factor Tu is present in mRNA-protein complexes

By two-dimensional gel electrophoresis, partial peptide mapping, and antibody binding we have shown that eukaryotic elongation factor Tu is in close contact with mRNA in rabbit reticulocytes. It can be crosslinked to mRNA by irradiating both polysomes and 40-80 S mRNA-protein complexes with short-wave UV light. To our knowledge this is the first case in which a known translation factor has been shown to be associated with mRNA in native ribonucleoproteins.     

Structural features in aminoacyl-tRNAs required for recognition by elongation factor Tu

In bacterial polypeptide synthesis aminoacyl-tRNA (aa-tRNA) bound to elongation factor Tu (EF-Tu) and GTP is part of a crucial intermediate ribonucleoprotein complex involved in the decoding of messenger RNA. The conformation and topology as well as the affinity of the macromolecules in this ternary aa-tRNA X EF-Tu X GTP complex are of fundamental importance for the nature of the interaction of the complex with the ribosome. The structural elements of aa-tRNA required for interaction with EF-Tu and GTP and the resulting functional implications are presented here.     

Antisuppression by mutations in elongation factor Tu

Two slow-growing kirromycin-resistant Escherichia coli mutants with altered EF-Tu (Ap and Aa) were studied in vivo in strains with an inactive tufB gene. Mutant form Aa was isolated as an antisuppressor of the tyrT(Su3) nonsense suppressor, as described here. Ap, the tufA gene product of strain D2216 (from A. Parmeggiani), has previously been shown to give an increased GTPase activity. The slow cellular growth rates of both EF-Tu mutants are correlated with decreased translational elongation rates. Ap and Aa significantly decrease suppression levels of both nonsense and missense suppressor tRNAs [tyrT(Su3), trpT(Su9), glyT(SuAGA/G)], but have only little or no effect on misreading by wild-type tRNAs. A particular missense suppressor, lysT(SuAAA/G), which acts by virtue of partial mischarging as the result of an alteration in the amino acid stem, is not significantly affected by the EF-Tu mutations. The combination of tufA(Aa) and a rpsD12 ribosomal mutation is lethal at room temperature and the double-mutant strain has an elevated temperature optimum (42 degrees C) for growth rate, translation rate and nonsense suppression. Our data indicate an alterated interaction between Aa and the ribosome, consistent with our in vitro results.     

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.     

Intrinsic fluorescence of elongation factor Tu in its complexes with GDP and elongation factor Ts

The intrinsic fluorescence properties of elongation factor Tu (EF-Tu) in its complexes with GDP and elongation factor Ts (EF-Ts) have been investigated. The emission spectra for both complexes are dominated by the tyrosine contribution upon excitation at 280 nm whereas excitation at 300 nm leads to exclusive emission from the single tryptophan residue (Trp-184) of EF-Tu. The fluorescence lifetime of this tryptophan residue in both complexes was investigated by using a multifrequency phase fluorometer which achieves a broad range of modulation frequencies utilizing the harmonic content of a mode-locked laser. These results indicated a heterogeneous emission with major components near 4.8 ns for both complexes. Quenching experiments on both complexes indicated limited accessibility of the tryptophan residue to acrylamide and virtually no accessibility to iodide ion. The quenching patterns exhibited by EF-Tu-GDP and EF-Tu X EF-Ts were, however, different; both quenchers were more efficient at quenching the emission from the EF-Tu x EF-Ts complex. Steady-state and dynamic polarization measurements revealed limited local mobility for the tryptophan in the EF-Tu x GDP complex whereas formation of the EF-Tu x EF-Ts complex led to a dramatic increase in this local mobility.     

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.     

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.     

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)     

Preparation of nucleotide-free elongation factor Tu and its stabilization by the antibiotic kirromycin

A rapid, highly reproducible procedure for the preparation of nucleotide-free elongation factor Tu (EF-Tu) is described. A microscale gel filtration is performed in a two-step elution, which takes less than 30 min and allows the preparation of nanomole amounts of the factor. The nucleotide-free EF-Tu is unstable, as measured by its ability to bind GDP. However, it can be stabilized either by the presence of a residual contamination of GDP of at least 1%, in the absence of Mg²⁺, or by kirromycin. In the presence of the latter component, the nucleotide-free EF-Tu is stable over a long period of time, similarly to the EF-Tu· GDP complex. Both GDP and kirromycin promote the reactivation of partially inactivated nucleotide-free EF-Tu, as measured by GDP binding and GTPase activity.     

A study of a mutant elongation factor properties of E. coli HAK88 and its mutant elongation factor Tu.

Summary The E. coli chromosome contains two genes for elongation factor Tu, tufA (near the fusidic acid resistance marker) and tufB (near the rifampicin resistance marker). It has been discovered that the mutant E. coli K12 strain HAK88 bears a mutation in the tufB gene, which leads to the synthesis of a protein of increased acidity. To determine whether the mutation has altered the protein's function in peptide chain elongation, we have compared the reactivities of normal tufA EF-Tu and mutant tufB EF-Tu (purified together from HAK88) with the components of the AA-tRNA binding cycle. Normal tufA EF-Tu and mutant tufB EF-Tu are indistinguishable in their affinities for GDP, EF-Ts, and phe-tRNA, and differ only slightly in their affinities for ribosomes. Coupled with the results of a separate study showing the similarity of the normal tufA and tufB gene products, these experiments demonstrate that the mutation has not altered the function of tufB EF-Tu in peptide chain elongation. Contrary to the original report (Kuwano et al., 1974; J. Mol. Biol. 86, 689–698) the HAK88 strains we have examined no longer possess a temperature-sensitive EF-Ts. The growth rates of HAK88 strains resemble the parent HAK8 strain in their lack of tRNA dependence but unlike HAK8 show varying degrees of temperature sensitivity. We conclude that HAK88 contains a physically altered but functionally intact tufB EF-Tu. The mutation in tufB should be valuable for studying in vivo the control of expression of the genes for EF-Tu.     

Translational regulation by modifications of the elongation factor Tu

EF-Tu fromE. coli, one of the superfamily of GTPase switch proteins, plays a central role in the fast and accurate delivery of aminoacyl-tRNAs to the translating ribosome. An overview is given about the regulatory effects of methylation, phosphorlation and phage-induced cleavage of EF-Tu on its function. During exponential growth, EF-Tu becomes monomethylated at Lys⁵⁶ which is converted to Me₂Lys upon entering the stationary phase. Lys⁵⁶ is in the GTPase switch-1 regions (residues 49–62), a strongly conserved site involved in interactions with the nucleotide and the 5′ end of tRNA. Methylation was found to attenuate GTP hydrolysis and may thus enhance translational accuracy.In vivo 5–10% of EF-Tu is phosphorylated at Thr³⁸² by a ribosome-associated kinase. In EF-Tu-GTP, Thr³⁸² in domain 3 has a strategic position in the interface with domain 1; it is hydrogen-bonded to Glu¹¹⁷ that takes part in the switch-2 mechanism, and is close to the T-stem binding site of the tRNA, in a region known for many kirromycin-resistance mutations. Phosphorylation is enhanced by EF-Ts, but inhibited by kirromycin. In reverse, phosphorylated EF-Tu has an increased affinity for EF-Ts, does not bind kirromycin and can no longer bind aminoacyl tRNA. Thein vivo role of this reversibles modification is still a matter of speculation. T4 infection ofE. coli may trigger a phage-exclusion mechanism by activation of Lit, a host-encoded proteinase. As a result, EF-Tu is cleaved site-specifically between Gly⁵⁹-Ile⁶⁰ in the switch-1 region. Translation was found to drop beyond a minimum level. Interestingly, the identical sequence in the related EF-G appeared to remain fully intact. Although the Lit cleavage-mechanism may eventually lead to programmed cell death, the very efficient prevention of phage multiplication may be caused by a novel mechanisms ofin cis inhibition of late T4 mRNA translation. Presented at theSymposium on Regulation of Translation of Genetic Information by Protein Phosphorylation, 21st Congress of the Czechoslovak Society for Microbiology, Hradec Králové (Czech Republic), September 6–10, 1998.     

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.     

Both positional isomers of aminoacyl-tRNA's are bound by elongation factor Tu.

Six purified Escherichia coli and yeast tRNA's were converted to positionally defined tRNA's terminating in 2'- and 3'-deoxyadenosine; the modified (amino-acyl) tRNA's were compared for their abilities to bind to elongation factor Tu (EF-Tu) in the presence both of GTP and guanylylimidodiphosphate (GMP-P(NH)P). Formation of aminoacyl-tRNA . EF-Tu . guanine nucleotide ternary complexes was monitored by gel filtration on Sephadex G-100 and Ultrogel ACA 44 columns and also by measurement of the ability of the factor to diminish the rate of chemical hydrolysis of the aminoacyl-tRNA's. The apparent positional specificity of the factor was found to be affected substantially both by the choice of guanine nucleotide and gel filtration resin utilized, but not in any systematic fashion. Likewise, assay of ternary complex formation by diminution of the rate of chemical deacylation failed to reveal any consistent positional preference from one isoacceptor to another. It is worthy of note that each modified aminoacyl-tRNA tested did form a ternary complex with EF-Tu under each of the experimental conditions used for assay, but that in each case the difference in affinity of the factor for isomeric aminoacyl-tRNA's was less than that between either of the modified aminoacyl-tRNA's and the corresponding unmodified species. On the basis of the experiments performed, we conclude that (i) EF-Tu has remarkable conformation flexibility, possibly reflecting its physiological role in recognizing 20 tRNA isoacceptors and (ii) the factor has no obvious preference for a single positional isomer of aminoacyl-tRNA and it is not clear that any preference that might exist could be established convincingly using tRNA's terminating in 2'- and 3'-deoxyadenosine.