A kirromycin resistant elongation factor EF‐Tu from <i>Escherichia coli</i> contains a threonine instead of an alanine residue in position 375

Duisterwinkel, F. J.; Graaf, J.Martien de; Kraal, Barend; Bosch, L.

doi:10.1016/0014-5793(81)80894-0

Cited by 32 publications

(17 citation statements)

References 39 publications

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“…These data and those in [15,16] show that amino acid replacement at position 375 of the EF-Tu polypeptide chain not only affects the interaction with the antibiotic kirromycin but also that with GTP. Table 2 Apparent association and dissociation rate constants for the complex between EF-Tu from various strains and the guanine nucleotides GTP and GDP at 50 mM of NI-F 4 and 5°C change than that by Thr (R = -CH ), since \ OH EF-TUD2216 is more resistant to kirromycin (table 1) it has a higher affinity for GTP (tables 2,3B and it exhibits a stronger activation of its GTPase centre ( fig.l, table 3A) than EF-'I'ULBE2045.…”

Section: Discussionsupporting

confidence: 74%

“…It has been argued [ 15,16] that position 3 75 is most probably the only site of mutation in EF-TULBE2045. The same arguments may be raised for EF-TUD2216, but we are less sure in the latter case because of the type of mutagenesis applied.…”

Section: Discussionmentioning

confidence: 99%

“…The two EF-Tu species resistant to kirromycin (LBE2045 and D2216)have one mutation site in common: amino acid residue 375 of the polypeptide chain [ 15,16]. While the wild-type contains an alanine in that position [6], EF-TUD2216 contains a valine [16] and EF-TULBE2045 a threonine [15].…”

Section: Introductionmentioning

confidence: 99%

“…While the wild-type contains an alanine in that position [6], EF-TUD2216 contains a valine [16] and EF-TULBE2045 a threonine [15].…”

Section: Introductionmentioning

confidence: 99%

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

et al. 1982

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Section: Discussionsupporting

confidence: 74%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

“…While the wild-type contains an alanine in that position [6], EF-TUD2216 contains a valine [16] and EF-TULBE2045 a threonine [15].…”

Section: Introductionmentioning

confidence: 99%

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

et al. 1982

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“…3). A375T was previously identified as the E. coli tufA R mutation (Duisterwinkel et al 1981) and the Salmonella typhimurium tufA8 mutation (Tubulekas et al 1991). …”

Section: Suppression Of Streptomycin-resistance Phenotype Of H76r By mentioning

confidence: 99%

A signal relay between ribosomal protein S12 and elongation factor EF-Tu during decoding of mRNA

Gregory¹,

Carr²,

Dahĺberg³

2008

RNA

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Codon recognition by aminoacyl-tRNA on the ribosome triggers a process leading to GTP hydrolysis by elongation factor Tu (EF-Tu) and release of aminoacyl-tRNA into the A site of the ribosome. The nature of this signal is largely unknown. Here, we present genetic evidence that a specific set of direct interactions between ribosomal protein S12 and aminoacyl-tRNA, together with contacts between S12 and 16S rRNA, provide a pathway for the signaling of codon recognition to EF-Tu. Three novel amino acid substitutions, H76R, R37C, and K53E in Thermus thermophilus ribosomal protein S12, confer resistance to streptomycin. The streptomycin-resistance phenotypes of H76R, R37C, and K53E are all abolished by the mutation A375T in EF-Tu. A375T confers resistance to kirromycin, an antibiotic freezing EF-Tu in a GTPase activated state. H76 contacts aminoacyl-tRNA in ternary complex with EF-Tu and GTP, while R37 and K53 are involved in the conformational transition of the 30S subunit occurring upon codon recognition. We propose that codon recognition and domain closure of the 30S subunit are signaled through aminoacyl-tRNA to EF-Tu via these S12 residues.

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

Noort¹,

Duisterwinkel²,

Jonák³

et al. 1982

The EMBO Journal

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The interaction of the polypeptide chain elongation factor Tu (EF‐Tu) with the antibiotic kirromycin and tRNA has been studied by measuring the extent of protein modification with N‐tosyl‐L‐phenylalanine chloromethylketone (TPCK) and N‐ethylmaleimide (NEM). Kirromycin protects both EF‐Tu.GDP and EF‐Tu.GTP against modification with TPCK. Binding of aminoacyl‐tRNA added at increasing concentrations to a solution of 40 microM EF‐Tu.GDP.kirromycin complex re‐exposes the TPCK target site on the protein. However, when the aminoacyl‐tRNA concentration is raised beyond 20 microM, TPCK labeling drops again and is blocked completely at approximately 300 microM aminoacyl‐tRNA. By contrast, addition of uncharged tRNA or N‐ acetylaminoacyl ‐tRNA enhances TPCK labeling of the protein over the entire tRNA concentration range studied. These data strongly suggest that kirromycin induces in EF‐Tu.GDP an additional tRNA binding site that can bind uncharged tRNA, aminoacyl‐tRNA, and N‐ acetylaminoacyl ‐tRNA. Support for this assumption is provided by measuring the modification of EF‐Tu.GDP with the sulfhydryl reagent NEM. Moreover, NEM modification also indicates an additional tRNA binding site on EF‐Tu.GTP.kirromycin, which could not be detected with TPCK. Mapping of the tryptic peptides of EF‐Tu.GDP labeled with [14C]TPCK revealed only one target site for this agent, i.e., cysteine‐81. Modification occurred at the same site in the presence and in the absence of kirromycin and uncharged tRNA.(ABSTRACT TRUNCATED AT 250 WORDS)

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A kirromycin resistant elongation factor EF‐Tu from Escherichia coli contains a threonine instead of an alanine residue in position 375

Cited by 32 publications

References 39 publications

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

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

A signal relay between ribosomal protein S12 and elongation factor EF-Tu during decoding of mRNA

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

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