Analysis and crystallization of a 25 kDa C‐terminal fragment of cloned elongation factor Ts from<i>Escherichia coli</i>

Bøgestrand, Søren; Wiborg, Ove; Thirup, Søren; Nyborg, Jens

doi:10.1016/0014-5793(95)00597-3

Cited by 10 publications

(8 citation statements)

References 43 publications

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“…The N-terminal deletion mutant of E. coli EF-Ts is unable to stimulate the activity of EF-Tu in guanine nucleotide exchange or in poly(U)-directed polymerization (data not shown). This observation is in agreement with previous results showing that a proteolytic derivative of E. coli EF-Ts lacking the N-terminal domain is unable to stimulate the activity of EF-Tu (20). The N-terminal deletion mutant of EF-Ts mt is unable to bind E. coli EF-Tu (data not shown).…”

Section: Interaction Of E Coli and Mitochondrial Ef-ts With E Coli supporting

confidence: 93%

“…The value obtained (8.6 ϫ 10 10 ) indicates that EF-Ts mt binds to E. coli EF-Tu quite tightly. The K obs for E. coli EF-Ts was also measured, and the corresponding binding constant (K Ts ) was calculated ( (20) and the x-ray structure of the E. coli EF-Tu⅐Ts complex (14) shows that the N-terminal region of E. coli EF-Ts folds into an independent domain (Fig. 1).…”

Section: Interaction Of E Coli and Mitochondrial Ef-ts With E Coli mentioning

confidence: 99%

“…1). This region is essential for the ability of E. coli EF-Ts to stimulate guanine nucleotide exchange with EF-Tu (20). Sequence alignment indicates that there is significant homology between E. coli EF-Ts and EF-Ts mt in the N-terminal domain and it is likely that it will fold in a similar three-dimensional structure.…”

Section: Interaction Of E Coli and Mitochondrial Ef-ts With E Coli mentioning

confidence: 99%

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Role of Domains in Escherichia coli and Mammalian Mitochondrial Elongation Factor Ts in the Interaction with Elongation Factor Tu

Zhang

Sun

Spremulli

1997

Journal of Biological Chemistry

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Bovine mitochondrial elongation factor Ts (EF-Ts mt ) stimulates the activity of Escherichia coli elongation factor Tu (EF-Tu). In contrast, E. coli EF-Ts is unable to stimulate mitochondrial EF-Tu. EF-Ts mt forms a tight complex with E. coli EF-Tu governed by an association constant of 8.6 ؋ 10 10 . This value is 100-fold stronger than the binding constant for the formation of the E. coli EF-Tu⅐Ts complex. To test which domain of EF-Ts mt is important for its strong binding with EF-Tu, chimeras were made between E. coli EF-Ts and EF-Ts mt . Replacing the N-terminal domain of E. coli EF-Ts with that of EF-Ts mt increases its binding to E. coli EF-Tu 2-3-fold. Replacing the N-terminal domain of EF-Ts mt with the corresponding region of E. coli EF-Ts decreases its binding to E. coli EF-Tu ϳ4 -5-fold. A chimera consisting of the C-terminal half of E. coli EF-Ts and the N-terminal half of EF-Ts mt binds to E. coli EF-Tu as strongly as EF-Ts mt .A chimera in which Subdomain N of the core of EF-Ts is replaced by the corresponding region of EFTs mt binds E. coli EF-Tu ϳ25-fold more tightly than E. coli EF-Ts. Thus, the higher strength of the interaction between EF-Ts mt and EF-Tu can be localized primarily to a single subdomain.The classical model for the elongation cycle of protein biosynthesis was developed on observations made with Escherichia coli EF-Tu.

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Section: Interaction Of E Coli and Mitochondrial Ef-ts With E Coli supporting

confidence: 93%

Section: Interaction Of E Coli and Mitochondrial Ef-ts With E Coli mentioning

confidence: 99%

Section: Interaction Of E Coli and Mitochondrial Ef-ts With E Coli mentioning

confidence: 99%

See 1 more Smart Citation

Role of Domains in Escherichia coli and Mammalian Mitochondrial Elongation Factor Ts in the Interaction with Elongation Factor Tu

Zhang

Sun

Spremulli

1997

Journal of Biological Chemistry

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“…Several properties of these mutants are described including (i) their binding to E. coli EF-Tu, (ii) their abilities to promote guanine nucleotide exchange with E. coli EF-Tu and to stimulate poly(U)-directed polymerization with the bacterial factor, and (iii) their abilities to stimulate the activity of EF-Tu mt . (14,20). This region of E. coli EF-Ts consists of three helical segments (h1, h2, and h3) and can be aligned readily with the NH 2 -terminal domain of EF-Ts mt (Fig.…”

Section: Resultsmentioning

confidence: 99%

Roles of Residues in Mammalian Mitochondrial Elongation Factor Ts in the Interaction with Mitochondrial and Bacterial Elongation Factor Tu

Zhang

Spremulli

1998

Journal of Biological Chemistry

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The crystal structure of the complex between Escherichia coli elongation factors Tu and Ts (EF-Tu⅐Ts) and subsequent mutagenesis work have provided insights into the roles of a number of residues in E. coli EF-Ts in its interaction with EF-Tu. The corresponding residues in bovine mitochondrial EF-Ts (EF-Ts mt ) have been mutated. The abilities of the resulting EF-Ts mt derivatives to stimulate the activities of both E. coli and mitochondrial EF-Tu have been tested. Mutation of several residues in EF-Ts mt corresponding to amino acids important for the activity of E. coli EF-Ts has little or no effect on the activity of the mitochondrial factor, suggesting that these factors may use somewhat different mechanisms to promote guanine nucleotide exchange. In general, mutations that reduce the strength of the interaction between EF-Ts mt and E. coli EF-Tu increase the ability of EF-Ts mt to stimulate the activity of the bacterial factor. In contrast, these mutations tend to reduce the ability of EF-Ts mt to stimulate the activity of EFTu mt . For example, F19A/I20A and H176A derivatives of EF-Ts mt are as active as E. coli EF-Ts in simulating E. coli EF-Tu. However, these mutations significantly decrease the ability of EF-Ts mt to stimulate EF-Tu mt .

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“…This deletion, however, completely inaotivates the EF-Ts in respect to nucleotide exchange activity m d complex formation with EF-Tu. A similar carboxy-terminal fragment of E. coli EF-Ts is equally inactive (Bflgestrand et al, 1995). The dimerisation site of EF-Ts, therefore, is expected to involve mainly the carboxy-terminal part of the molecule.…”

mentioning

confidence: 99%

Elongation Factor Ts from Thermus thermophilus

Blank¹,

Nock²,

Kreutzer³

et al. 1996

European Journal of Biochemistry

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The gene encoding the elongation factor Ts from Thermus therrnophilus was sequenced, cloned and the protein overproduced in Escherichia coli. In comparison to the EF-Ts from E. coli with 282 amino acid residues, EF-Ts from 7: thermophilus is considerably shorter, differing by 86 amino acids. EF-Ts from the thermophile is stable at high temperatures, which facilitates its separation from E. coli proteins.Purified 7: thermophilus EF-Ts forms a homodimer with a disulfide bridge between the two cysteine residues at position 190. The modification of Cys190 by iodoacetamide affects neither the dimerization nor the ability of EF-Ts to facilitate the nucleotide exchange of elongation factor Tu. The disulfide bridge was detected only in purified EF-Ts, but not in protein extracts immediately after cell disruption. The physiological role of this disulfide bridge remains, therefore, unclear. Besides the quaternary (EF-Tu . EF-Ts), complex, a ternary EF-Tu . EF-Ts, complex was detected by gel permeation chromatography and polyacrylamide gel electrophoresis. Trypsin cleavage after Lys48 or modification of Cys78 yield inactive EF-Ts, that does not bind to EF-Tu but is still capable of forming homodimers.

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Analysis and crystallization of a 25 kDa C‐terminal fragment of cloned elongation factor Ts fromEscherichia coli

Cited by 10 publications

References 43 publications

Role of Domains in Escherichia coli and Mammalian Mitochondrial Elongation Factor Ts in the Interaction with Elongation Factor Tu

Role of Domains in Escherichia coli and Mammalian Mitochondrial Elongation Factor Ts in the Interaction with Elongation Factor Tu

Roles of Residues in Mammalian Mitochondrial Elongation Factor Ts in the Interaction with Mitochondrial and Bacterial Elongation Factor Tu

Elongation Factor Ts from Thermus thermophilus

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