Biomedical subjects

B Kraal

Publications and source records attributed to B Kraal.

At least 37 records · Page 2Linked to original sources

PubMed1995

Phosphorylation of elongation factor Tu prevents ternary complex formation.

The elongation factor Tu (EF-Tu) is a member of the GTP/GDP-binding proteins and interacts with various partners during the elongation cycle of protein biosynthesis thereby mediating the correct binding of amino-acylated transfer RNA (aa-tRNA) to the acceptor site (A-site) of the ribosome. After GTP hydrolysis EF-Tu is released in its GDP-bound state. In vivo, EF-Tu is post-translationally modified by phosphorylation. Here we report that the phosphorylation of EF-Tu by a ribosome associated kinase activity is drastically enhanced by EF-Ts. The antibiotic kirromycin, known to block EF-Tu function, inhibits the modification. This effect is specific, since kirromycin-resistant mutants do become phosphorylated in the presence of the antibiotic. On the other hand, phosphorylated wild-type EF-Tu does not bind kirromycin. Most interestingly, the phosphorylation of EF-Tu abolishes its ability to bind aa-tRNA. In the GTP conformation the site of modification is located at the interface between domains 1 and 3 and is involved in a strong interdomain hydrogen bond. Introduction of a charged phosphate group at this position will change the interaction between the domains, leading to an opening of the molecule reminiscent of the GDP conformation. A model for the function of EF-Tu phosphorylation in protein biosynthesis is presented.

Anti-Bacterial Agents↗

PubMed1995

Substitution of Arg230 and Arg233 in Escherichia coli elongation factor Tu strongly enhances its pulvomycin resistance.

Pulvomycin is a strong inhibitor of protein synthesis, known to prevent the binding of aminoacyl-tRNA to elongation factor Tu.GTP (EF-Tu.GTP). Recently, three pulvomycin-resistant mutant strains have been isolated by targeted mutagenesis of the tufA gene resulting in EF-Tu substitutions at positions 230, 333 or 334. In order to analyze the functions of arginine residues located in domain II, with respect to pulvomycin resistance and the interaction with aminoacyl-tRNA, we have investigated the effect of the substitutions of the highly conserved residues Arg230 and Arg233 by site-directed mutagenesis. We have purified two mutants species, [R233S]EF-TuHis and [R230V, R233F]EF-TuHis, both with a C-terminal histidine extension to enable purification by Ni2+ affinity chromatography. In this study, we describe the in vitro characterization of these mutant proteins. The results show that the concomitant substitution of residues at positions 230 and 233, dramatically increases the pulvomycin resistance. Preliminary evidence is presented that protein synthesis is inhibited by an EF-Tu.GDP.pulvomycin complex rather than by EF-Tu.GTP.pulvomycin. Moreover, the mutant [R230V, R233F]EF-TuHis shows a stronger protection of the ester bond of aminoacyl-tRNA than wild-type EF-Tu.

Aminoglycosides↗

PubMed1994

The structural and functional basis for the kirromycin resistance of mutant EF-Tu species in Escherichia coli.

A structural and functional understanding of resistance to the antibiotic kirromycin in Escherichia coli has been sought in order to shed new light on the functioning of the bacterial elongation factor Tu (EF-Tu), in particular its ability to act as a molecular switch. The mutant EF-Tu species G316D, A375T, A375V and Q124K, isolated by M13mp phage-mediated targeted mutagenesis, were studied. In this order the mutant EF-Tu species showed increasing resistance to the antibiotic as measured by poly(U)-directed poly(Phe) synthesis and intrinsic GTPase activities. The K'd values for kirromycin binding to mutant EF-Tu.GTP and EF-Tu.GDP increased in the same order. All mutation sites cluster in the interface of domains 1 and 3 of EF-Tu.GTP, not in that of EF-Tu.GDP. Evidence is presented that kirromycin binds to this interface of wild-type EF-Tu.GTP, thereby jamming the conformational switch of EF-Tu upon GTP hydrolysis. We conclude that the mutations result in two separate mechanisms of resistance to kirromycin. The first inhibits access of the antibiotic to its binding site on EF-Tu.GTP. A second mechanism exists on the ribosome, when mutant EF-Tu species release kirromycin and polypeptide chain elongation continues.

Anti-Bacterial Agents↗

PubMed1994

Synergism between the GTPase activities of EF-Tu.GTP and EF-G.GTP on empty ribosomes. Elongation factors as stimulators of the ribosomal oscillation between two conformations.

A remarkable positive cooperativity between the GTPase activities of EF-Tu and EF-G on empty ribosomes from Escherichia coli has been discovered. This cooperativity implies a decrease of the corresponding apparent KM values of the empty ribosome for either elongation factor: from more than 10 microM to 0.5 microM for EF-Tu.GTP by the addition of 0.25 microM EF-G and from 0.7 microM to 0.5 microM for EF-G.GTP by the addition of 3 microM EF-Tu. In a further analysis of this phenomenon, the effects of various specific antibiotics were studied: thiostrepton, fusidic acid, tetracycline, pulvomycin and kirromycin appeared to inhibit the synergistic effect, whereas streptomycin was found to stimulate it. Even in the present minimal system the ribosomes respond to the above-mentioned antibiotics in a way surprisingly similar to that in the coupled system with mRNA and tRNAs. The cooperativity seems not to be due to a simultaneous binding of the two elongation factors to the ribosome as revealed by studying the effects of fusidic acid and kirromycin, and by band-shift experiments by means of gel electrophoresis under non-denaturing conditions. Our experimental data and the kinetic analysis of alternative models provide evidence that EF-Tu.GTP and EF-G.GTP interact sequentially with empty ribosomes that oscillate between two different conformations, one for each elongation factor. Apparently, ribosomes have an intrinsic property for oscillation as normally observed during protein synthesis with a frequency paced by the events of tRNA binding and translocation.

Allosteric Regulation↗

PubMed1993

Divergent effects of fluoroaluminates on the peptide chain elongation factors EF-Tu and EF-G as members of the GTPase superfamily.

Fluoraluminates are thought to mimic the gamma-phosphate of GTP and thus, together with GDP, perturb the functioning of heterotrimeric GTP-binding G-proteins. Here we show they do inhibit the ribosome-stimulated GTPase activity of EF-G from Escherichia coli via the formation of a stable complex with EF-G-GDP and ribosomes. In contrast, no perturbed interactions were observed in a similar ribosomal complex with EF-Tu. Interestingly, in the absence of ribosomes both EF-Tu an EF-G remain totally unaffected by fluoraluminates. For members of the GTPase superfamily such differential effects have not been described before.

1-Propanol↗

PubMed1993

Elongation factors in protein synthesis.

Recent discoveries of elongation factor-related proteins have considerably complicated the simple textbook scheme of the peptide chain elongation cycle. During growth and differentiation the cycle may be regulated not only by factor modification but also factor replacement. In addition, rare tRNAs may have their own rare factor proteins. A special case is the acquisition of resistance by bacteria to elongation factor-directed antibiotics. Pertinent data from the literature and our own work with Escherichia coli and Streptomyces are discussed. The GTP-binding domain of EF-Tu has been studied extensively, but little molecular detail is available on the interactions with its other ligands or effectors, or on the way they are affected by the GTPase switch signal. A growing number of EF-Tu mutants obtained by ourselves and others are helping us in testing current ideas. We have found a synergistic effect between EF-Tu and EF-G in their uncoupled GTPase reactions on empty ribosomes. Only the EF-G reaction is perturbed by fluoroaluminates.

Animals↗

PubMed1992

Isolation and functional analysis of histidine-tagged elongation factor Tu.

The study of the structure/function relationships of the Escherichia coli elongation factor Tu (EF-Tu) via mutagenesis has been hampered by difficulties encountered in separating the mutated factor from other proteins, in particular native EF-Tu. Here we describe a novel system for the purification of EF-Tu mutant species, based on metal-ion affinity chromatography. To facilitate rapid and efficient purification we designed a recombinant EF-Tu with an additional C-terminal sequence of one serine and six histidine residues. A cell extract containing the His-tagged EF-Tu (EF-TuHis) is applied to a Ni(2+)-nitrilotriacetic acid column. EF-TuHis can be selectively eluted with an imidazole containing buffer, yielding a preparation of more than 95% purity, free of wild-type EF-Tu. In-vitro and in-vivo functional analyses show that EF-TuHis resembles the wild-type EF-Tu, which makes this one-step isolation procedure a promising tool for the study of the interactions of mutant EF-Tu with the various components of the elongation cycle. The new isolation procedure was successfully applied for the purification of a mutant EF-TuHis with a Glu substitution for Lys237, a residue possibly involved in the binding of aminoacyl-tRNA.

Amino Acid Sequence↗

PubMed1991

Kirromycin drastically reduces the affinity of Escherichia coli elongation factor Tu for aminoacyl-tRNA.

We have studied the interaction between EF-Tu-GDP or EF-Tu-GTP in complex with kirromycin or aurodox (N1-methylkirromycin) and aminoacyl-tRNA, N-acetylaminoacyl-tRNA, or deacylated tRNA. Three independent methods were used: zone-interference gel electrophoresis, GTPase stimulation, and fluorescence. All three methods revealed that kirromycin induces a severe drop in the stability of the complex of EF-Tu-GTP and aminoacyl-tRNA of about 3 orders of magnitude. The affinities of EF-Tu-kirromycin-GTP and EF-Tu-kirromycin-GDP for aa-tRNA were found to be of about the same order of magnitude. We conclude that kirromycin and related compounds do not induce a so-called GTP-like conformation of EF-Tu with respect to tRNA binding. The findings shed new light on the mechanism of action of the antibiotic during the elongation cycle. In contrast to indirect evidence previously obtained in our laboratory [Van Noort et al. (1982) EMBO J. 1, 1199-1205; Van Noort et al. (1986) Proc. Natl. Acad. Sci. U.S.A. 71, 4910-4914], we were unable to demonstrate complexes of EF-Tu-aurodox-GTP/GDP with N-acetylaminoacyl-tRNA or deacylated tRNA by direct detection using zone-interference gel electrophoresis. Modification with N-tosyl-L-phenylalanine chloromethyl ketone (TPCK) decreases the affinity of EF-Tu-kirromycin-GTP for aminoacyl-tRNA, just like it does in the absence of the antibiotic.

Electrophoresis, Agar Gel↗

PubMed1991

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.

Electrophoresis, Polyacrylamide Gel↗

PubMed1990

Fluoroaluminates do not affect the guanine-nucleotide binding centre of the peptide chain elongation factor EF-Tu.

EF-Tu is often referred to as a model for guanine-nucleotide-binding regulatory proteins (G-proteins), since X-ray diffraction analysis of its GTP-binding domain shows a detailed location of the 'consensus' amino acid sequences involved in nucleotide binding. Fluoroaluminates are thought to mimick the gamma-phosphate in the GTPase centre on account of their activating effect on a variety of GTP binding proteins. In the case of EF-Tu, we could find no such effects on the basis of at least three independent functional assays. We did notice, however, complicating interactions between free nucleotides, fluoroaluminates and other ligands. By consequence, if indeed AlF4- behaves as a gamma-phosphate analogue in G-proteins, then EF-Tu must have a different GDP/GTP binding site, despite of the conserved consensus sequences.

Aluminum↗

PubMed1990

The interaction between aminoacyl-tRNA and the mutant elongation factors Tu AR and B0.

The binding of Tyr-[AEDANS-s2C]tRNA(Tyr) (Tyr-tRNA(Tyr) modified at the penultimate cytidine residue with a thio group at position 2 of the pyrimidine ring, to which an N-(acetylaminoethyl)-5-naphthylamine-1-sulfonic acid fluorescence group is attached) to mutant elongation factor (EF)-Tu species from E. coli, EF-TuAR (Ala-375----Thr) and EF-TuBO (Gly-222----Asp), both complexed to GTP, was investigated in absence of kirromycin by measuring the change in fluorescence of the modified tRNA induced by complex formation. The calculated dissociation constant in the case of EF-TuAR is about 4 nM and in the case of EF-TuB0, about 1 nM. These values are higher than that of wild-type EF-Tu, which was 0.24 nM measured with the same system. The affinity between either EF-TuB0.kirromycin.GDP or EF-TuB0.kirromycin.GTP on the one hand, and a mixture of aminoacyl-tRNAs on the other, was measured with zone-interference gel electrophoresis. The dissociation constants are 20 microM and 7 microM, respectively, a factor of about two higher than in the case of wild-type EF-Tu.kirromycin. These findings provide a clue for the observed increase in translational errors in strains carrying the mutations. Furthermore, the experiments with EF-TuB0.kirromycin deepen our understanding of the effects of the B0 mutation on the kirromycin phenotype of the mutant cells concerned.

Anti-Bacterial Agents↗

PubMed1988

Zone-interference gel electrophoresis: a new method for studying weak protein-nucleic acid complexes under native equilibrium conditions.

A new and general electrophoresis method is described for the determination of dissociation constants of weak macromolecular complexes in the range of 10(-6) to 10(-4) M. The method is based on the measurement of the migration distance of a macromolecular complex in rapid dynamic equilibrium as a function of the interacting ligand concentration in a surrounding zone. Special advantages of the method are: its high sensitivity (dependent on the autoradiography, immunoblotting or staining technique used), its speed (electrophoresis time 20 min), and the independence of the Kd determination on the sample concentration of macromolecules. The latter is of great value for labile macromolecules: unknown partial inactivation does not influence the measurement. Studying the interactions between elongation factor EF-Tu and tRNA from E. coli we found for EF-Tu.GTP.aurodox.aminocyl-tRNA a Kd of 3 microM and for EF-Tu.GDP.aurodox.aminoacyl-tRNA a Kd of 11 microM at 9 degrees C.

Anti-Bacterial Agents↗

PubMed1987

Effects of the mutation glycine-222----aspartic acid on the functions of elongation factor Tu.

We have studied the properties of a mutant elongation factor Tu, encoded by tufB (EF-TuBo), in which Gly-222 is replaced by Asp. For its purification from the kirromycin-resistant EF-Tu encoded by tufA (EF-TuAr), a method was developed by exploiting the different affinities to kirromycin of the two factors and the competition between kirromycin and elongation factor Ts (EF-Ts) for binding to EF-Tu. The resulting EF-TuBo kirromycin and EF-TuAr EF-Ts complexes are separated by chromatography on diethylaminoethyl-Sephadex A-50. For the first time we have succeeded in obtaining a tufB product in homogeneous form. Compared with wild-type EF-Tu, EF-TuBo displays essentially the same affinity for GDP and GTP, with only the dissociation rate of EF-Tu GTP being slightly faster. Protection of amino-acyl-tRNA (aa-tRNA) against nonenzymatic deacylation by different EF-Tu species indicates that conformational alterations occur in the ternary complex EF-TuBo GTP aa-tRNA. However, the most dramatic modification is found in the EF-TuBo interaction with the ribosome. Its activity in poly(Phe) synthesis as well as in the GTPase activity associated with the interaction of its ternary complex with the ribosome mRNA complex requires higher Mg2+ concentrations than wild-type EF-Tu (Mg2+ optimum at 10-14 vs. 6 mM), even if EF-TuBo can sustain enzymatic binding of aa-tRNA to ribosomes at low Mg2+. The anomalous behavior of EF-TuBo is reflected in a remarkable increase of the fidelity in poly(Phe) synthesis, especially at high Mg2+ concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)

Aspartic Acid↗

PubMed1986

Methylation in vivo of elongation factor EF-Tu at lysine-56 decreases the rate of tRNA-dependent GTP hydrolysis.

In this paper we show, that the in vivo methylation of the elongation factor Tu from Escherichia coli is correlated with the growth phase of the bacterium. Methylation occurs at one position only, i.e. Lys-56, and initially results in monomethylation during logarithmic growth. Upon entering the stationary phase of E. coli, monomethyllysine is gradually converted into dimethyllysine. We have undertaken an extensive comparison between the properties of the highly methylated EF-Tu and unmodified EF-Tu. No gross conformational differences, as measured by the rate of mild tryptic cleavage, were observed. The dissociation rates of the nucleotides GDP and GTP appear likewise to be unaffected by the methylation, just as is the stimulatory effect of the elongation factor Ts upon these rates. Whereas tRNA binding at the classical binding site of EF-Tu (site I) also appears not to be affected by the methylation of the protein, tRNA binding at site II is. Although the apparent affinity of tRNA for site II remains unaltered upon methylation of EF-Tu, the conformational effects of tRNA binding at this site become different. Both the GTPase activity of the protein and the reactivity of Cys-81 are significantly less stimulated by the tRNA when EF-Tu is methylated. A possible physiological implication of this phenomenon is discussed.

Escherichia coli↗

PubMed1986

GTPase center of elongation factor Tu is activated by occupation of the second tRNA binding site.

Interaction of the elongation factor EF-Tu with the antibiotic kirromycin results in activation of the GTPase center of the factor and in induction of an additional tRNA binding site (tRNA binding site II to distinguish it from the classical tRNA binding site I). Activation of the GTPase center under these conditions is stimulated by addition of tRNA. Two-fold evidence is presented that this stimulation is due to tRNA binding to site II rather than to site I. First, a strong correlation is observed between stimulation of the GTPase activity and enhancement of the reactivity of Cys-81 of EF-Tu toward N-ethylmaleimide at various concentrations of aminoacyl-tRNA, deacylated tRNA, and N-acetylaminoacyl-tRNA. The latter effects signal tRNA binding to site II. Stimulation of the kirromycin-induced GTPase activity by tRNA binding to the factor also occurs when binding to site I is completely abolished. Such an abolishment was achieved by treating EF-Tu extensively with the thiol reagent L-1-tosylamido-2-phenylethyl chloromethyl ketone. EF-Tu X GTP thus treated has lost its ability to protect the ester bond of aminoacyl-tRNA. The relevance of these data for the sequence of events during protein synthesis and for control of translational fidelity is discussed.

Allosteric Regulation↗

PubMed1985

Mutants of the elongation factor EF-Tu, a new class of nonsense suppressors.

Read-through of nonsense codons has been studied in wild-type Escherichia coli cells and in cells harbouring mutant species of the elongation factor EF-Tu. The two phenomena differ essentially. Readthrough of UGA in wild-type cells is reduced by inactivation of tufB but is restored to the original level by introducing into the cell plasmid-borne EF-Tu. This shows that the natural UGA leakiness is dependent on the intracellular concentration of EF-Tu. Strains of E. coli harbouring mutant species of the elongation factor EF-Tu suppress the nonsense codons UAG, UAA and UGA. Suppression shows a codon context dependence. It requires the combined action of two different EF-Tu species: EF-TuAR(Ala 375----Thr) and EF-TuBo(Gly 222----Asp). Cells harbouring EF-TuAR(Ala 375----Thr) and wild-type EF-TuB, or wild-type EF-TuA and EF-TuBo(Gly 222----Asp) do not display suppressor activity. These data demonstrate that mutated tuf genes form an additional class of nonsense suppressors. The requirement for two different mutant EF-Tu species raises the question whether translation of sense codons also occurs by the combined action of two EF-Tu molecules on the ribosome.

Base Sequence↗

PubMed1985

A second tRNA binding site on elongation factor Tu is induced while the factor is bound to the ribosome.

Previously, we reported that the antibiotic kirromycin induces two tRNA-binding sites on the elongation factor Tu. The classical binding site (site I) binds aminoacyl-tRNA and, with much less affinity, deacylated tRNA. The kirromycin-induced site II binds aminoacyl-tRNA, peptidyl-tRNA, and deacylated tRNA with comparable affinities. Accordingly, 3'-oxidized tRNA can be cross-linked in the presence of the antibiotic to two specific sites of EF-Tu: Lys-237 and Lys-208. Here, we report that 3'-oxidized tRNAPhe, bound to a ribosome-poly(U) complex, can also be cross-linked to either one of these two sites. When located in the ribosomal peptidyl site, it cross-links exclusively to Lys-208; when located in the ribosomal aminoacyl site, it cross-links exclusively to Lys-237, irrespective of the presence of kirromycin. Since no cross-linking could be detected in the absence of ribosomes and kirromycin, we conclude that the tRNA-binding site II is induced upon interaction of aminoacyl-tRNA-EF-Tu-GTP with the ribosome-mRNA complex. The results indicate that, on the ribosome, EF-Tu interacts with peptidyl-site-bound peptidyl-tRNA through tRNA-binding site II and with aminoacyl-site-bound aminoacyl-tRNA through tRNA-binding site I.

Binding Sites↗

PubMed1984

Specific alterations of the EF-Tu polypeptide chain considered in the light of its three-dimensional structure.

Specific alterations of the elongation factor Tu (EF-Tu) polypeptide chain have been identified in a number of mutant species of this elongation factor. In two species, Ala-375, located on domain II, was found by amino acid analysis to be replaced by Thr and Val, respectively. These replacements substantially lower the affinity of EF-Tu.GDP for the antibiotic kirromycin. Since kirromycin can be cross-linked to Lys-357, also located on domain II but structurally very far from Ala-375, these data suggest that the replacements alter the relative position of domains I and II. The Ala-375 replacements also lower the dissociation rates of the binary complexes EF-Tu.GTP and the binding constants for EF-Tu.GTP and Phe-tRNA. It is conceivable that these effects are also mediated by movements of domains I and II relative to each other. Replacement of Gly-222 by Asp has been found in another mutant by DNA sequence analysis of the cloned tufB gene, coding for this mutant EF-Tu. Gly-222 is part of a structural domain, characteristic for a variety of nucleotide binding enzymes. Its replacement by Asp does not abolish the ability of EF-Tu to sustain protein synthesis. It increases the dissociation rate of EF-Tu.GTP by approximately 30%. In the presence of kirromycin this mutant species of EF-Tu.GDP does not bind to the ribosome, in contrast to its wild-type counterpart. A possible explanation is now open for experimental verification.

Amino Acids↗