Interchangeability of elongation factor‐Tu and elongation factor‐1 in aminoacyl‐tRNA binding to 70 S and 80 S ribosomes

Grasmuk, Hans; Nolan, Robert D.; Drews, Jürgen

doi:10.1016/0014-5793(77)80592-9

Cited by 13 publications

(6 citation statements)

References 10 publications

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“…The vast majority of data describing the interaction of elongation factors with the ribosome come from studies done in prokaryotes. For example, it has been shown that the eukaryotic translocase eEF2 does not function with the prokaryotic 70S ribosome and conversely the prokaryotic EF-G is not functional in the context of the 80S ribosome (Grasmuk et al, 1977 ;Taira et al, 1972). In a recent study, Uchiumi et al (1999) swapped the L8 complex consisting of L7\L12 and L10 proteins in E. coli with its counterpart from rat, comprising P1, P2 and P0 proteins, and showed that the hybrid ribosome displayed eEF2-dependent but not EF-G-dependent GTPase activity.…”

Section: Discussionmentioning

confidence: 99%

Species-specific inhibition of fungal protein synthesis by sordarin: identification of a sordarin-specificity region in eukaryotic elongation factor 2 The GenBank accession numbers for the sequences reported in this manuscript are AF107286–AF107291, AF292693 and AF248644.

Shastry

Nielsen

et al. 2001

Microbiology

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The sordarin class of natural products selectively inhibits fungal protein synthesis by impairing the function of eukaryotic elongation factor 2 (eEF2). Mutations in Saccharomyces cerevisiae eEF2 or the ribosomal stalk protein rpP0 can confer resistance to sordarin, although eEF2 is the major determinant of sordarin specificity. It has been shown previously that sordarin specifically binds S. cerevisiae eEF2 while there is no detectable binding to eEF2 from plants or mammals, despite the high level of amino acid sequence conservation among these proteins. In both whole-cell assays and in vitro translation assays, the efficacy of sordarin varies among different species of pathogenic fungi. To investigate the basis of sordarin's fungal selectivity, eEF2 has been cloned and characterized from several sordarin-sensitive and -insensitive fungal species. Results from in vivo expression of Candida species eEF2s in S. cerevisiae and in vitro translation and growth inhibition assays using hybrid S. cerevisiae eEF2 proteins demonstrate that three amino acid residues within eEF2 account for the selectivity of this class of compounds. It is also shown that the corresponding residues at these positions in human eEF2 are sufficient to confer sordarin insensitivity to S. cerevisiae identical to that observed with mammalian eEF2.

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Section: Discussionmentioning

confidence: 99%

Species-specific inhibition of fungal protein synthesis by sordarin: identification of a sordarin-specificity region in eukaryotic elongation factor 2 The GenBank accession numbers for the sequences reported in this manuscript are AF107286–AF107291, AF292693 and AF248644.

Shastry

Nielsen

et al. 2001

Microbiology

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“…It is generally recognized that the eukaryotic translocase EF-2 does not function with the prokaryotic 70 S ribosome, and the prokaryotic factor EF-G is not accessible to the eukaryotic 80 S ribosome (36,37). The present study demonstrates that replacement of the stalk protein complex L10-L7/L12 (L8 complex) in E. coli ribosome with rat counterpart P0-P1-P2 (P complex) changes the factor binding specificity from EF-G (homologous) to EF-2 (heterologous).…”

Section: Discussionmentioning

confidence: 99%

Replacement of L7/L12.L10 Protein Complex in Escherichia coli Ribosomes with the Eukaryotic Counterpart Changes the Specificity of Elongation Factor Binding

Uchiumi¹,

Hori²,

Nomura³

et al. 1999

Journal of Biological Chemistry

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The L8 protein complex consisting of L7/L12 and L10 in Escherichia coli ribosomes is assembled on the conserved region of 23 S rRNA termed the GTPase-associated domain. We replaced the L8 complex in E. coli 50 S subunits with the rat counterpart P protein complex consisting of P1, P2, and P0. The L8 complex was removed from the ribosome with 50% ethanol, 10 mM MgCl 2 , 0.5 M NH 4 Cl, at 30°C, and the rat P complex bound to the core particle. Binding of the P complex to the core was prevented by addition of RNA fragment covering the GTPase-associated domain of E. coli 23 S rRNA to which rat P complex bound strongly, suggesting a direct role of the RNA domain in this incorporation. The resultant hybrid ribosomes showed eukaryotic translocase elongation factor (EF)-2-dependent, but not prokaryotic EF-G-dependent, GTPase activity comparable with rat 80 S ribosomes. The EF-2-dependent activity was dependent upon the P complex binding and was inhibited by the antibiotic thiostrepton, a ligand for a portion of the GTPase-associated domain of prokaryotic ribosomes. This hybrid system clearly shows significance of binding of the P complex to the GTPase-associated RNA domain for interaction of EF-2 with the ribosome. The results also suggest that E. coli 23 S rRNA participates in the eukaryotic translocase-dependent GTPase activity in the hybrid system. Binding of translocases, EF1 -G⅐GTP in prokaryotes and EF-2⅐GTP in eukaryotes, to a specific site of the ribosome causes GTP hydrolysis that drives translocation of peptidyl-tRNA from the A-site to the P-site during protein biosynthesis (1-3). The binding site of the prokaryotic EF-G has been identified on the universally conserved regions, the "GTPase-associated domain" surrounding residue 1067 and the "sarcin/ricin loop" of residues 2653-2667 in 23 S rRNA (4, 5). Interaction of the eukaryotic EF-2 with the equivalent domains of 28 S rRNA has been also suggested (6 -8). Despite the highly conserved features of the two RNA domains which interact with the translocases EF-G and EF-2, these factors are not interchangeable between prokaryotic and eukaryotic translational systems.Another component, long implicated in the ribosomal interaction with the translocase, is the stalk protein L7/L12 in prokaryotic ribosomes (9 -11). This protein together with L10 forms a stable pentameric complex, (L7/L12)2(L7/L12)2L10, termed L8 (12), and this complex binds to the GTPase-associated domain of 23 S rRNA through its interaction with L10 (13,14). The eukaryotic counterpart of the prokaryotic L8 is the "P complex" consisting of homodimers of P1 and P2 and monomeric P0 (15, 16). Involvement of these constituent proteins in the activity of eukaryotic EF-2 has been demonstrated in vitro (17,18). An essential role of protein P0 for assembly of the complex into yeast ribosomes and for cell viability has also been shown in vivo (19,20). Rat P complex reconstituted from isolated P1, P2, and P0 specifically binds to the GTPase-associated domain of 28 S rRNA, probably through P0 protein (21, 22). ...

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“…GDP, EF-Ts and EF-G were a gift from H. Grasmuk. Their preparation and the analysis of their purity have been described [13].…”

Section: Preparation Of Elongation Factorsmentioning

confidence: 99%

The Effects of Tiamulin, a Semisynthetic Pleuromutilin Derivative, on Bacterial Polypeptide Chain Initiation

Dornhelm

Högenauer

1978

Eur J Biochem

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Tiamulin, a water-soluble and highly effective semisynthetic derivative of pleuromutilin leads to the formation of physiologically inactive polypeptide chain initiation complexes which readily decompose and do not enter the phase of peptide chain elongation. Once elongation has begun it continues even in the presence of tiamulin as has been shown by measuring the formation of N-acetylphenylalanine-poly(phenyla1anine). The formation of abortive initiation complexes was observed regardless of whether AcPhe-tRNA of Met-tRNA was used as an initiator or whether artificial messengers or a natural messenger, like R17 bacteriophage RNA, was used. When this drug was acting on whole cells, it led to the disappearance of polysomes. The only structures which could be detected were of the monosome size. Therefore, polysomes seem to elongate the polypeptide chains in whole cells in the presence of this antibiotic, but since effective reinitiation is blocked, the polysome pool of the cell soon becomes depleted.Pleuromutilin, a tricyclic diterpene antibiotic which is produced by a basidiomycete, is active against bacteria and mycoplasms. When the glycolic acid residue of the natural product is replaced by basic thio-ether side chains the antibacterial activity is increased considerably. One semisynthetic compound with these chemical characteristics, the 14-deoxy-14-[(2-diethylaminoethy1)mercapto-acetoxy]mutilin hydrogen fumarate, has been given the generic name 'tiamulin'. The formulae of both the natural substance and the semisynthetic compound were described in previous communications from this institute [l, 21. In order to elucidate the mode of action of pleuromutilin and its derivatives, tiamulin and chemically closely related substances have been selected as model compounds. By experiments described in an earlier communication [2] these pleuromutilin derivatives were characterized as inhibitors of prokaryotic ribosomes. At micromolar concentrations they cause a dramatic decline in the poly(pheny1alanine) synthesis, the synthesis of formylmethionine-puromycin or that of the initial dipeptide of the bacteriophage R17 coat protein. In the latter reaction it appeared as if the formation of initiation complexes was only slightly affected, while the binding of alanyl-tRNA to the ribosomal A-site was inhibited to a large extent. Elongation of nascent polypeptides on natural polysomes, when assayed in vitro, showed little sensitivity towards tiamulin. The formation of the first peptide bond, involving the initiating formylmethionine residue, thus appears to be particularly sensitive towards pleuromutilin derivatives.In order to explain this sensitivity of the first peptide bond, the present work was undertaken. The result showed that although initiation complexes are formed in the presence of the drug, they are defective and therefore do not allow further propagation of the protein chain.Abbreviations. AcPhe-poly(Phe), N-acetylphenylalanine-poly-(phenylalanine); EF-T, mixture of elongation factors EF-Tu and EF-Ts; Hepes, 4-(2-hy...

show abstract

Interchangeability of elongation factor‐Tu and elongation factor‐1 in aminoacyl‐tRNA binding to 70 S and 80 S ribosomes

Cited by 13 publications

References 10 publications

Species-specific inhibition of fungal protein synthesis by sordarin: identification of a sordarin-specificity region in eukaryotic elongation factor 2 The GenBank accession numbers for the sequences reported in this manuscript are AF107286–AF107291, AF292693 and AF248644.

Species-specific inhibition of fungal protein synthesis by sordarin: identification of a sordarin-specificity region in eukaryotic elongation factor 2 The GenBank accession numbers for the sequences reported in this manuscript are AF107286–AF107291, AF292693 and AF248644.

Replacement of L7/L12.L10 Protein Complex in Escherichia coli Ribosomes with the Eukaryotic Counterpart Changes the Specificity of Elongation Factor Binding

The Effects of Tiamulin, a Semisynthetic Pleuromutilin Derivative, on Bacterial Polypeptide Chain Initiation

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