Allosteric changes of the guanine nucleotide site of elongation factor EF‐Tu

Swart, Guido W.M.; Kraal, B.; Bosch, L.; Parmeggiani, Andrea

doi:10.1016/0014-5793(82)80228-7

Cited by 24 publications

(13 citation statements)

References 18 publications

(31 reference statements)

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“…EF-Tu (Lb) has a considerably higher affinity than EF-Tu (Ec) for GTP. In this respect, the protein is similar to that of kirromycin-resistant mutants of E. coli [16]. …”

Section: Heat Stabilitymentioning

confidence: 72%

Kirromycin‐resistant elongation factor Tu from wild‐type of Lactobacillus brevis

Wörner

Wolf

1982

FEBS Letters

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Properties of the elongation factor Tu from Lactobacillus brevis which is naturally insensitive to kirromycin are described. The protein is characterized by an unusual nucleotide‐binding site with increased affinity for GTP and extreme heat stability. EF‐Tu is sensitive to pulvomycin in the assay of polyphenylalanine synthesis. However, the failure of the protein to display pulvomycin‐dependent GDP‐binding and GTPase activity indicates that pulvomycin action in L. brevis differs from that in E. coli.

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“…EF-Tu (Lb) has a considerably higher affinity than EF-Tu (Ec) for GTP. In this respect, the protein is similar to that of kirromycin-resistant mutants of E. coli [16]. …”

Section: Heat Stabilitymentioning

confidence: 72%

Kirromycin‐resistant elongation factor Tu from wild‐type of Lactobacillus brevis

Wörner

Wolf

1982

FEBS Letters

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“…It is germane to mention that in the absence of effectors influencing its interaction with GDP/GTP, the initial rate of the singleturnover GTPase of EF-Tu-GTP (0.06 mmol of GTP hydrolyzed per sec per mol of EF-Tu) is very close to the rate of the multiple-round GTPase of the G domain (see above). In the case of EF-Tu the multiple turnover hydrolysis is strongly inhibited by the tightly bound product GDP (28,29). The absolute specificity of EF-Tu for guanine nucleotides is conserved in the G domain; no detectable ATPase activity was found when tested using [y-32P]ATP of very high specific activity (data not shown).…”

Section: Resultsmentioning

confidence: 97%

Properties of a genetically engineered G domain of elongation factor Tu.

Parmeggiani¹,

Swart²,

Mortensen³

et al. 1987

Proc. Natl. Acad. Sci. U.S.A.

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The G domain of elongation factor Tu (EFTu), representing the N-terminal half of the factor according to its three-dimensional model traced at high resolution, has been isolated by genetic manipulation of tufA and purified to homogeneity. The G domain, whose primary structure shares homology with the eukaryotic protein p21, is capable of supporting the basic activities of the intact molecule (guanine nucleotide binding in 1:1 molar ratio and GTPase activity).However, it is no longer exposed to the allosteric mechanisms regulating EF-Tu. The G-domain complexes with GTP and GDP display similar Kd values in the ,uM range, in contrast to EF-Tu that binds GDP much more tightly than GTP. Its GTPase shows the characteristics of a slow turnover reaction The guanine nucleotide-binding proteins have attracted much attention, particularly since a number of proteins regulating different cell functions (proliferation, hormone response, neurotransmission, and protein biosynthesis) were found to belong to this family of proteins (for example, see refs. 1-3). Elongation factor Tu (EF-Tu) from Escherichia coli is one of the best characterized guanine nucleotide-binding proteins (4, 5) and the only one whose three-dimensional structure has been partially elucidated at high resolution (6, 7). Its 393-amino acid polypeptide chain is folded in three distinct domains. The N-terminal domain (residues 1-200) binding GDP or GTP has primary structure features common to the ras protein p21 and other nucleotide-binding enzymes (6)(7)(8)(9)(10)(11). Of the three domains of EF-Tu, the N domain displays an a/l3 type structure, traced at high resolution (0.27-0.29 nm), consisting of six a-helices alternating with six f3-strands, typical for the class of nucleotide-binding proteins (6, 7). Fig.

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“…The evidence available shows that mutant factors resistant to factortargeted drugs may differ from susceptible factors merely by one amino acid. In fact, substitutions of alanine at position 375 within the allosterically important kirromycin-binding domain of E. coli EF-Tu result in kirromycin resistance (19). We note, however, that the basic phylogenetic stability of the drug-sensitive phenotype within the eubacterial kingdom indicates that the site involved in kirromycin binding is subject to severe selective constraints.…”

Section: Resultsmentioning

confidence: 80%

Unique antibiotic sensitivity of archaebacterial polypeptide elongation factors

Londei¹,

Sanz²,

Altamura³

et al. 1986

J Bacteriol

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The antibiotic sensitivity of the archaebacterial factors catalyzing the binding of aminoacyl-tRNA to ribosomes (elongation factor Tu [EF-Tu] for eubacteria and elongation factor 1 [EF1] for eucaryotes) and the translocation of peptidyl-tRNA (elongation factor G [EF-GI for eubacteria and elongation factor 2 [EF2] for eucaryotes) was investigated by using two EF-Tu and EF1 [EF-Tu(EF1)]-targeted drugs, kirromycm and pulvomycin, and the EF-G and EF2 [EF-G(EF2)]-targeted drug fusidic acid. The interaction of the inhibitors with the target factors was monitored by using polyphenylalanine-synthesizing cell-free systems. A survey of methanogenic, halophilic, and sulfur-dependent archaebacteria showed that elongation factors of organisms belonging to the methanogenic-halophilic and sulfur-dependent branches of the "third kingdom" exhibit different antibiotic sensitivity spectra. Namely, the methanobacterial-halobacterial EF-Tu(EFl)-equivalent protein was found to be sensitive to pulvomycin but insensitive to kirromycin, whereas the methanobacterialhalobacterial EF-G(EF2)-equivalent protein was found to be sensitive to fusidic acid. By contrast, sulfurdependent thermophiles were unaffected by all three antibiotics, with two exceptions; Thermococcus celer, whose EF-Tu(EF1)-equivalent factor was blocked by pulvomycin, and Thermoproteus tenax, whose EF-G(EF2)-equivalent factor was sensitive to fusidic acid. On the whole, the results revealed a remarkable intralineage heterogeneity of elongation factors not encountered within each of the two reference (eubacterial and eucaryotic) kingdoms.The two classically recognized lines of cellular descent, eubacteria and eucaryotes, are each endowed with a specific set of polypeptide chain elongation factors whose component parts usually can only cooperate with ribosomes of their own lineage (14, 18). Within each lineage, however, factors and ribosomes are functionally exchangeable for in vitro polypeptide synthesis (5).Elongation factors and ribosomes of the recently discovered archaebacteria (7) appear to define a third class of functional specificity encompassing organisms as taxonomically diverse as methane-producing (Methanococcus vannielii) and sulfur-dependent (Thermoplasma acidophilum) archaebacteria (13); that is, reciprocal combinations of factors and ribosomes from distant archaebacterial sources are active in polypeptide synthesis, but archaebacterial factors are functionally restricted to ribosomes of their own lineage.The aim of the present study was to establish whether intralineage distinctions among archaebacterial elongation factors could be detected on structural grounds. For this purpose, factors from a variety of archaebacteria covering most known divisions of the "third kingdom" were probed for the presence or absence of specific sites which, in eubacteria and eucaryotes, interact with factor-targeted inhibitors of protein synthesis. This approach also provides insight into the degree of structural relatedness among functionally homologous factors of the three ki...

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Allosteric changes of the guanine nucleotide site of elongation factor EF‐Tu

Cited by 24 publications

References 18 publications

Kirromycin‐resistant elongation factor Tu from wild‐type of Lactobacillus brevis

Kirromycin‐resistant elongation factor Tu from wild‐type of Lactobacillus brevis

Properties of a genetically engineered G domain of elongation factor Tu.

Unique antibiotic sensitivity of archaebacterial polypeptide elongation factors

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