Inhibition by thiopeptin of ribosomal functions associated with T and G factors

Kinoshita, Tadatoshi; Liou, Yei-Fei; Tanaka, Nobuo

doi:10.1016/0006-291x(71)90790-x

Cited by 50 publications

(19 citation statements)

References 10 publications

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“…Nevertheless, taken together with the observations that thiostrepton inhibits the activities of both factors (9,10) and that a single ribosomal protein is essential for these activities (23) *, my results make the single binding site the most convincing interpretation.…”

Section: Resultssupporting

confidence: 52%

“…Among many unsolved questions, it is not known whether there are one or several GTPase enzymes on the ribosome, or whether the elongation factors are themselves GTPases. Recent reports that treatment of ribosomes with the antibiotic thiostrepton inhibits both EF G-and EF Tcatalyzed GTP hydrolysis (9)(10)(11) support the proposal that both factors interact at the same site on the ribosome. If we consider that the substrates for the site, aminoacyl-tRNA-EF Tu-GTP and EF G + GTP, appear to be widely different in structure and function, this is a surprising conclusion.…”

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confidence: 82%

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Elongation Factors EF Tu and EF G Interact at Related Sites on Ribosomes

Miller

1972

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

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The binding of elongation factor EF G to ribosomes inhibits the subsequent reaction of the ribosomes with the ternary complex aminoacyl-tRNA EF Tu GTP. Both the hydrolysis of GTP and the binding of aminoacyl-tRNA to ribosomes are nearly abolished by the previous binding of factor EF G to ribosomes in the presence of either fusidic acid plus either GTP or a nonhydrolyzable analog of GTP. The results suggest that each elongation factor binds to the same region on the ribosome. The GTPase activities of both factors EF G and EF Tu may be activated by interaction at the same ribosomal site, as has been previously suggested by others.In the process of protein biosynthesis, the hydrolysis of GTP is required for the addition of each succeeding amino acid to a growing peptide (for a recent review, see ref 1). During the elongation stage, GTP participates with elongation factor EF Tu in the binding of aminoacyl-tRNA to the ribosome as part of the intermediate aminoacyl-tRNA*EF Tu GTP complex (2, 3). When this complex interacts with the ribosome, GTP is hydrolyzed, aminoacyl-tRNA is bound to the ribosome, and EF Tu GDP is released (4, 5). After peptide bond formation, hydrolysis of GTP, catalyzed by factor EF G, is required for ejection of the deacylated tRNA (6) and for the translocation of the ribosome along the message (7, 8). Thus, two molecules of GTP appear to be required for the addition of each amino acid.The role of GTP hydrolysis in these reactions remains unclear. Among many unsolved questions, it is not known whether there are one or several GTPase enzymes on the ribosome, or whether the elongation factors are themselves GTPases. Recent reports that treatment of ribosomes with the antibiotic thiostrepton inhibits both EF G-and EF Tcatalyzed GTP hydrolysis (9-11) support the proposal that both factors interact at the same site on the ribosome. If we consider that the substrates for the site, aminoacyl-tRNA-EF Tu-GTP and EF G + GTP, appear to be widely different in structure and function, this is a surprising conclusion. Since it is possible that thiostrepton might inhibit two independent sites, a direct test was made to see whether one of the factors inhibited the activity of the other.To this end, experiments were undertaken to see whether binding factor EF G to the ribosome inhibited the hydrolysis of GTP in Phe-tRNA EF Tu GTP (referred to here as the "ternary complex"). These studies show that factor EF GI when bound to ribosomes in the presence of either the nonhydrolyzable GTP analogue guanylyl-5'-methylenediphosphonate (GMPPCP) (12), or fusidic acid plus GTP (13)

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Section: Resultssupporting

confidence: 52%

mentioning

confidence: 82%

Elongation Factors EF Tu and EF G Interact at Related Sites on Ribosomes

Miller

1972

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

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“…Scolnick and Caskey previously demonstrated (27) (27) It has been shown previously (6)(7)(8)(9)(10)(11)(12)(13)30) that several partial reactions of protein synthesis that involve ribosomal proteins L7 and L12 are also inhibited by thiostrepton. Table 4 inhibited by thiostrepton at concentrations giving complete inhibition of codon-directed release.…”

Section: Resultsmentioning

confidence: 93%

“…These same reactions are also inhibited by the antibiotic thiostrepton (6)(7)(8)(9)(10)(11)(12). In addition, as initially suggested by Hamel et al (1), L7 and L12 are also required for the enzymatic binding of fMet-tRNA to ribosomest (13).…”

mentioning

confidence: 97%

The Requirement for Ribosomal Proteins L7 and L12 in Peptide-Chain Termination

Brot¹,

Tate

Caskey

et al. 1974

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

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Proteins L7 and L12 from 50S ribosomal subunits of Escherichia coli are required for peptidechain termination. This termination process is inhibited by thiostrepton. Since both thiostrepton-treated ribosomes and those depleted of L7 and L12 have a markedly reduced ability to form release factor UA[3H]A ribosome complexes, the binding of release factors to the ribosome appears to be the primary site of inhibition.The ribosomal proteins L7 and L12 of Escherichia coli have been shown to be involved in the elongation factor Tu (EF-Tu)-dependent binding of aminoacyl-tRNA to ribosomes (1, 2) and in the elongation factor G (EF-G)-dependent hydrolysis of GTP (3-5). These same reactions are also inhibited by the antibiotic thiostrepton (6)(7)(8)(9)(10)(11)(12). In addition, as initially suggested by Hamel et al. (1), L7 and L12 are also required for the enzymatic binding of fMet-tRNA to ribosomest (13). Since these reactions all involve hydrolysis of the -y-phosphate of GTP and since L7 and L12 do not affect ribosomal peptidyl transferase activity (1), it appears that L7 and L12 might be required for reactions involving GTP hydrolysis dependent on a soluble factor and the 50S ribosomal subunit. The hydrolysis of GTP is required for peptide-chain termination in vitro, as studied with reticulocyte release factor (RF) and ribosomes (14). Although no such requirement has been demonstrated for peptide-chain termination in E. coli in vitro, a protein factor, RF-3, and guanine nucleotides are known to influence the ribosomal binding of E. coli termination factors RF-1 and RF-2 (15). Recently the binding of EF-G to E. coli ribosomes, which requires L7 and L12, was shown to inhibit the interaction of RF-1 and RF-2 with ribosomes (16). For these reasons the effects of the ribosomal proteins L7 and L12 and the antibiotic thiostrepton were studied with respect to their influence on peptide-chain termination. The present report provides evidence that L7 and L12 are required for peptidechain termination in E. coli and that this process is inhibited by thiostrepton. MATERIALS AND METHODSMaterials. Ammonium chloride-washed ribosomes were isolated from E. coli B as described (17), and ribosomal subunits were prepared from these ribosomes by the procedure of Pestka and Nirenberg (18). The ribosomal proteins L7 and L12 were extracted from the 50S ribosomal subunits with an Abbreviations: L7 and L12, 50S ribosomal proteins according to the nomenclature of Kaltschmidt and Wittmann (38); release factors, RF-1, RF-2, and RF-3; elongation factor Tu, EF-Tu; elongation factor G, EF-G.

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“…Phe-tRNA (11) were formed and then isolated by sucrose gradient centrifugation as indicated in legend of Phe-tRNAPhe. Because bf the very high synthetic activity shown by the purified complexes, these experiments have to be performed in presence of ATP and phenylalanyl-tRNA synthetase in order to maintain a linear rate of incorporation in the two following conditions : (a) the two complexes are mixed and then polypeptide synthesis is started by adding a rate-limiting amount of EF-G (random distribution or free exchange of EF-G between the two complexes); (b) after one complex has reached its maximum rate of synthesis in the presence of the given amount of EF-G the second one is added (dynamic exchange of EF-G from complex I to complex 11).…”

Section: Pig6 Exchange Of Eli'-a! During Ply(phenyla1anine) Synthesmentioning

confidence: 99%

Properties of the Elongation Factors from Escherichia coli

Chinali

Parmeggiani

1973

European Journal of Biochemistry

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The interaction of elongation factor G (EF‐G) with ribosomes has been studied during or in the absence of elongation of the polypeptide chain in a system from Escherichia coli in vitro. Mutual saturation curves of ribosomes and EF‐G suggest a rapid exchange of the factor from ribosome to ribosome. In the GTPase reaction, V/2 was reached at [ribosomes]/[EF‐G] ∼ 14 and at [EF‐G]/[ribosomes] ∼ 33. In the poly(U)‐directed poly(phenylalanine) synthesis, V/2 was obtained at [ribosomes]/[EF‐G] ∼ 12 and at [EF‐G]/[ribosomes] ∼ 6. Kinetics of the exchange of EF‐G between two pure acetyl‐aminoacyl‐tRNA ·mRNA · ribosome complexes containing synthetic homo‐ or heteropolymeric mRNAs were investigated by following their respective EF‐G‐dependent incorporation of differently labelled amino acids into the corresponding polypeptides in experimental conditions in which EF‐G was either added free or already prebound to one of the complexes prior to addition of the second one. In all experiments the rates of incorporation showed that the exchange of EF‐G between the two complexes was immediate and complete. These observations indicate that EF‐G behaves as a virtually free component which interacts catalytically with ribosomes during each round of elongation of the polypeptide chain.

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Inhibition by thiopeptin of ribosomal functions associated with T and G factors

Cited by 50 publications

References 10 publications

Elongation Factors EF Tu and EF G Interact at Related Sites on Ribosomes

Elongation Factors EF Tu and EF G Interact at Related Sites on Ribosomes

The Requirement for Ribosomal Proteins L7 and L12 in Peptide-Chain Termination

Properties of the Elongation Factors from Escherichia coli

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