Construction of a fully active Cys-less elongation factor Tu: Functional role of conserved cysteine 81

Laurentiis, Evelina Ines De; Mo, Fan; Wieden, Hans-Joachim

doi:10.1016/j.bbapap.2011.02.007

Cited by 16 publications

(39 citation statements)

References 31 publications

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“…Furthermore, mutation of Cys-81 in EF-Tu of E. coli revealed that replacement of Cys-81 with glycine impaired the ability of EF-Tu to bind both aminoacyltRNA and nucleotides (36). Another study of mutations in EF-Tu of E. coli demonstrated that replacement of Cys-81 with either serine or methionine did not affect the affinity of nucleotides for EF-Tu but decreased the affinity of aminoacyl-tRNA for binding to EF-Tu (16). By contrast, replacement with alanine had no obvious effects on the binding affinity of nucleotides and aminoacyl-tRNA for EF-Tu (16).…”

Section: Inactivation Of Ef-tu Is Due To the Oxidation Of A Cysteinementioning

confidence: 99%

“…Another study of mutations in EF-Tu of E. coli demonstrated that replacement of Cys-81 with either serine or methionine did not affect the affinity of nucleotides for EF-Tu but decreased the affinity of aminoacyl-tRNA for binding to EF-Tu (16). By contrast, replacement with alanine had no obvious effects on the binding affinity of nucleotides and aminoacyl-tRNA for EF-Tu (16). In view of the limited variability at the site of the cysteine residue, De Laurentiis et al (16) proposed that the side chain of Cys-81 might not interact directly with either nucleotides or aminoacyl-tRNA but might play a role in stabilizing the ternary complex that consists of EF-Tu, GTP, and aminoacyl-tRNA.…”

Section: Inactivation Of Ef-tu Is Due To the Oxidation Of A Cysteinementioning

confidence: 99%

“…In analyses of bacteria, this particular cysteine residue was found in 111 (89%) of 125 aligned sequences, and substitution by alanine and methionine was found in only 13 (10%) and 1 (1%) sequence, respectively (16).…”

Section: Inactivation Of Ef-tu Is Due To the Oxidation Of A Cysteinementioning

confidence: 99%

“…Role of Cys-82 in EF-Tu-Alignment of the deduced amino acid sequences of EF-Tu from various organisms revealed that Cys-82 is strongly conserved in the EF-Tu of several species of cyanobacteria, prokaryotes, and plants (16,35). In analyses of bacteria, this particular cysteine residue was found in 111 (89%) of 125 aligned sequences, and substitution by alanine and methionine was found in only 13 (10%) and 1 (1%) sequence, respectively (16).…”

Section: Inactivation Of Ef-tu Is Due To the Oxidation Of A Cysteinementioning

confidence: 99%

“…A clue to the understanding of the mechanism of oxidation of EF-Tu is provided by the presence of a specific cysteine residue that is strongly conserved in various species of bacteria (15,16). EF-Tu of Synechocystis includes a single cysteine residue, namely, Cys-82, and this residue corresponds to the residue that is strongly conserved.…”

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confidence: 99%

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Oxidation of a Cysteine Residue in Elongation Factor EF-Tu Reversibly Inhibits Translation in the Cyanobacterium Synechocystis sp. PCC 6803

Yutthanasirikul¹,

Nagano²,

Jimbo³

et al. 2016

Journal of Biological Chemistry

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Translational elongation is susceptible to inactivation by reactive oxygen species (ROS) in the cyanobacterium Synechocystis sp. PCC 6803, and elongation factor G has been identified as a target of oxidation by ROS. In the present study we examined the sensitivity to oxidation by ROS of another elongation factor, EF-Tu. The structure of EF-Tu changes dramatically depending on the bound nucleotide. Therefore, we investigated the sensitivity to oxidation in vitro of GTP-and GDP-bound EF-Tu as well as that of nucleotide-free EF-Tu. Assays of translational activity with a reconstituted translation system from Escherichia coli revealed that GTP-bound and nucleotide-free EF-Tu were sensitive to oxidation by H 2 O 2 , whereas GDPbound EF-Tu was resistant to H 2 O 2 . The inactivation of EF-Tu was the result of oxidation of Cys-82, a single cysteine residue, and subsequent formation of both an intermolecular disulfide bond and sulfenic acid. Replacement of Cys-82 with serine rendered EF-Tu resistant to inactivation by H 2 O 2 , confirming that Cys-82 was a target of oxidation. Furthermore, oxidized EF-Tu was reduced and reactivated by thioredoxin. Gel-filtration chromatography revealed that some of the oxidized nucleotide-free EF-Tu formed large complexes of >30 molecules. Atomic force microscopy revealed that such large complexes dissociated into several smaller aggregates upon the addition of dithiothreitol. Immunological analysis of the redox state of EF-Tu in vivo showed that levels of oxidized EF-Tu increased under strong light. Thus, resembling elongation factor G, EF-Tu appears to be sensitive to ROS via oxidation of a cysteine residue, and its inactivation might be reversed in a redox-dependent manner.

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Section: Inactivation Of Ef-tu Is Due To the Oxidation Of A Cysteinementioning

confidence: 99%

Section: Inactivation Of Ef-tu Is Due To the Oxidation Of A Cysteinementioning

confidence: 99%

Section: Inactivation Of Ef-tu Is Due To the Oxidation Of A Cysteinementioning

confidence: 99%

Section: Inactivation Of Ef-tu Is Due To the Oxidation Of A Cysteinementioning

confidence: 99%

mentioning

confidence: 99%

See 3 more Smart Citations

Oxidation of a Cysteine Residue in Elongation Factor EF-Tu Reversibly Inhibits Translation in the Cyanobacterium Synechocystis sp. PCC 6803

Yutthanasirikul¹,

Nagano²,

Jimbo³

et al. 2016

Journal of Biological Chemistry

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Elongation Factor Tu Switch I Element is a Gate for Aminoacyl-tRNA Selection

Girodat

Blanchard

Wieden

et al. 2020

Journal of Molecular Biology

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Labeled EF-Tus for Rapid Kinetic Studies of Pretranslocation Complex Formation

Liu¹,

Kavaliauskas²,

Schrader

et al. 2014

ACS Chem. Biol.

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The universally conserved translation elongation factor EF-Tu delivers aminoacyl(aa)-tRNA in the form of an aa-tRNA·EF-Tu·GTP ternary complex (TC) to the ribosome where it binds to the cognate mRNA codon within the ribosomal A-site, leading to formation of a pretranslocation (PRE) complex. Here we describe preparation of QSY9 and Cy5 derivatives of the variant E348C-EF-Tu that are functional in translation elongation. Together with fluorophore derivatives of aa-tRNA and of ribosomal protein L11, located within the GTPase associated center (GAC), these labeled EF-Tus allow development of two new FRET assays that permit the dynamics of distance changes between EF-Tu and both L11 (Tu-L11 assay) and aa-tRNA (Tu-tRNA assay) to be determined during the decoding process. We use these assays to examine: (i) the relative rates of EF-Tu movement away from the GAC and from aa-tRNA during decoding, (ii) the effects of the misreading-inducing antibiotics streptomycin and paromomycin on tRNA selection at the A-site, and (iii) how strengthening the binding of aa-tRNA to EF-Tu affects the rate of EF-Tu movement away from L11 on the ribosome. These FRET assays have the potential to be adapted for high throughput screening of ribosomal antibiotics.

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Construction of a fully active Cys-less elongation factor Tu: Functional role of conserved cysteine 81

Cited by 16 publications

References 31 publications

Oxidation of a Cysteine Residue in Elongation Factor EF-Tu Reversibly Inhibits Translation in the Cyanobacterium Synechocystis sp. PCC 6803

Oxidation of a Cysteine Residue in Elongation Factor EF-Tu Reversibly Inhibits Translation in the Cyanobacterium Synechocystis sp. PCC 6803

Elongation Factor Tu Switch I Element is a Gate for Aminoacyl-tRNA Selection

Labeled EF-Tus for Rapid Kinetic Studies of Pretranslocation Complex Formation

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