The elongation factor Tu.kirromycin complex has two binding sites for tRNA molecules.

Noort, Johannes M. van; Duisterwinkel, F. J.; Jonák, Jiřı́; Sedláček, Juraj; Kraal, Barend; Bosch, L.

doi:10.1002/j.1460-2075.1982.tb00013.x

Cited by 31 publications

(20 citation statements)

References 30 publications

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“…It can be concluded, in accordance with our previous report, that the affinity of deacylated tRNA for binding site II is significantly lower than that of aminoacyl-tRNA. Dependence of the modification on the concentration of N-acetylaminoacyl-tRNA is somewhat different from that reported earlier (13). Presumably, deacylation during storage of the previous preparation may account for this difference.…”

Section: Methodscontrasting

confidence: 91%

“…Bulk tRNA was almost completely aminoacylated with a mixture of all of the amino acids (13) and was stored in 2 mM KOAc (pH 5.0) at -80°C. Immediately prior to use, it was dialyzed against reaction buffer.…”

Section: Methodsmentioning

confidence: 99%

“…The reaction was allowed to proceed at 0C for 30 min, after which it was stopped by the addition of 2-mercaptoethanol to a final concentration of 10 mM and 100 tug of bovine serum albumin as a carrier. The mixtures were subsequently precipitated with 3 ml of 7% (wt/vol) trichloroacetic acid, filtered on Whatman GF/C filters, and analyzed by scintillation spectrometry (13).…”

Section: Methodsmentioning

confidence: 99%

“…Another effect of kirromycin is the induction of an additional tRNA binding site (site II) on EF-Tu, essentially different from the classical site (13). Cross-linking of the 3'-terminal adenosine of tRNA to EF-Tu indicated that the two sites are topographically distinct (14).…”

mentioning

confidence: 99%

“…This question is highly relevant since the induction of site II and the activation of the GTPase center also occur when EF-Tu interacts with the ribosome as a ternary complex during the elongation cycle. Moreover, site II can bind peptidyl-tRNA (13), and tRNA cross-linking to EF-Tu on the ribosome revealed that tRNA positioned at the ribosomal peptidyl site (P site) binds to site II, whereas tRNA positioned at the ribosomal aminoacyl site (A site) binds to site I (15).…”

mentioning

confidence: 99%

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

Noort¹,

Kraal²,

Bosch³

1986

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

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GTPase center of elongation factor Tu is activated by occupation of the second tRNA binding site (GTP February 20, 1986 ABSTRACT 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-acetylaminoacyltRNA. The latter effects signal tRNA binding to site II. Stimulation of the kirromycin-induced GTPase activity by

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Section: Methodscontrasting

confidence: 91%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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

Noort¹,

Kraal²,

Bosch³

1986

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

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

Duisterwinkel¹,

Kraal²,

Graaf³

et al. 1984

The EMBO Journal

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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.

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

Vijgenboom¹,

Vink²,

Kraal³

et al. 1985

The EMBO Journal

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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.

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The elongation factor Tu.kirromycin complex has two binding sites for tRNA molecules.

Cited by 31 publications

References 30 publications

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

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

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

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

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