The Elongation Phase of Protein Synthesis

Czworkowski, John; Moore, Peter B.

doi:10.1016/s0079-6603(08)60366-9

Cited by 61 publications

(36 citation statements)

References 179 publications

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“…Stabilization of the EF2-Ribosome Complex-During the translocation cycle, GTP is bound by the EF2-ribosome complex, followed by an extremely rapid hydrolysis to EF2-GDP, and a conformational change that releases EF2 for the next round of translocation (13). Fusidic acid is a universal EFG and EF2 inhibitor that inhibits translocation by stabilizing the EF2-GDP-ribosome complex (14).…”

Section: Resultsmentioning

confidence: 99%

Elongation Factor 2 as a Novel Target for Selective Inhibition of Fungal Protein Synthesis

Justice¹,

Hsu²,

Tse³

et al. 1998

Journal of Biological Chemistry

182

138

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Elongation factor 2 (EF2) is an essential protein catalyzing ribosomal translocation during protein synthesis and is highly conserved in all eukaryotes. It is largely interchangeable in translation systems reconstituted from such divergent organisms as human, wheat, and fungi. We have identified the sordarins as selective inhibitors of fungal protein synthesis acting via a specific interaction with EF2 despite the high degree of amino acid sequence homology exhibited by EF2s from various eukaryotes. In vitro reconstitution assays using purified components from human, yeast, and plant cells demonstrate that sordarin sensitivity is dependent on fungal EF2. Genetic analysis of sordarin-resistant mutants of Saccharomyces cerevisiae shows that resistance to the inhibitor is linked to the genes EFT1 and EFT2 that encode EF2. Sordarin blocks ribosomal translocation by stabilizing the fungal EF2-ribosome complex in a manner similar to that of fusidic acid. The fungal specificity of the sordarins, along with a detailed understanding of its mechanism of action, make EF2 an attractive antifungal target. These findings are of particular significance due to the need for new antifungal agents.The elongation phase of translation in fungi requires the soluble elongation factors EF1␣, EF2, and EF3. EF1␣ and EF2 are members of the GTPase superfamily of proteins and are characterized by common structural motifs and their ability to alternate between conformational states in response to binding GDP or GTP. These proteins are required for translation in all eukaryotes, while EF3 is unique to fungi and essential for fungal protein synthesis (1). EF2 catalyzes the translocation of the ribosome along messenger RNA, presumably by stimulating a gross rearrangement of the ribosome that results in peptidyl-tRNA transfer and the movement of mRNA by one codon. The protein sequence of EF2 has been highly conserved throughout evolution, with Saccharomyces cerevisiae EF2 sharing 66% identity and 85% homology to human EF2. Despite this high degree of similarity, a class of tetracyclic diterpene glycoside natural products, the sordarins, has now been identified as selective inhibitors of EF2 function in fungal protein synthesis. Sordarin, produced by species of the fungal genus Sordaria, was described as an antifungal agent in 1970 (2, 3), but the mode of action of this family has not been examined until now. In this report, we show that sordarins specifically bind to the S. cerevisiae EF2-ribosome complex and block protein synthesis by inhibiting the release of EF2 from the posttranslocational ribosome. Our observations show that it is possible to inhibit fungal EF2 specifically, which may provide an opportunity for developing antifungal agents with a unique and selective mechanism of action. EXPERIMENTAL PROCEDURESSordarin was isolated essentially as described for Sordaria arenosa (2). Reticulocyte and wheat germ lysates were obtained from Promega.Assays-IC 50 values were determined from growth inhibition assays in which cells were inoculated a...

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Section: Resultsmentioning

confidence: 99%

Elongation Factor 2 as a Novel Target for Selective Inhibition of Fungal Protein Synthesis

Justice¹,

Hsu²,

Tse³

et al. 1998

Journal of Biological Chemistry

182

138

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“…In our models, we assume that tRNAs maintain their crystallographically determined conformation when bound to the ribosome+ It is possible that tRNAs undergo significant conformational change when bound to the ribosome as suggested by the crystal structures of tRNA-synthetase complexes (Rould et al+, 1989;Ruff et al+, 1991) and by low-resolution cryoelectron microscopy difference maps of tRNA-ribosome complexes (Malhotra et al+, 1998)+ To understand the molecular mechanism of translation, it is essential to elucidate the mutual arrangement of tRNAs on the ribosome+ So far, at least a half-dozen different binding states of tRNA have been described (Green & Noller, 1997)+ In these studies, the tRNAs are bound in the A/A and P/P states, corresponding to the classical A and P sites+ They are in the S orientation with the P tRNA on the left and A tRNA on the right as viewed from the 30S toward the 50S subunit (Fig+ 7A)+ The anticodon stem-loops are at the bottom, interacting mainly with the 30S subunit and mRNA; the 39-CCA ends are at the top, interacting with the 50S subunit+ Although the 39 termini of the two tRNAs are insufficiently close to allow peptide bond formation, flexibility of their single-stranded 39-ACCA termini could allow a closer approach+ Placement of A-site tRNA on the right is consistent with the interaction of the EF-Tu ternary complex near the L7/L12 stalk at the right of the 50S subunit (Girshovich et al+, 1986;Stark et al+, 1997b)+ Also, in the crystal structure of the EF-Tu:GDPNP:tRNA ternary complex, the T-loop side of the A-site tRNA is in contact with EF-Tu (Nissen et al+, 1995)+ Therefore, this side cannot face P tRNA, consistent with the S orientation+ An important ribosomal function is translocation, the coordinated movement of the tRNA-mRNA complex within the ribosome following peptide bond formation (Kaziro, 1978;Spirin, 1985;Czworkowski & Moore, 1996;Wilson & Noller, 1998)+ During translocation, tRNAs move from right to left from A site to P site as shown in Figure 7A+ Our model predicts that translocation of A-site tRNA into the P site could be accomplished by a rotational movement of about 458 around an axis drawn from the 39-CCA end through the anticodon stemloop of the A-site tRNA, coupled with a translational movement of about 24+5 Å from right to left+ Interestingly, we do not detect any cleavages in the anticodon stem-loop region of P tRNA, consistent with the previous observation that the anticodon stem-loop of P tRNA is protected from hydroxyl radicals by the 30S subunit (Hüttenhofer & Noller, 1994), most likely by features of 16S rRNA that line the cleft of the 30S subunit+ Nor do we detect any cleavages in the 39-CCA end of P tRNA from Fe(II) tethered to the 59 terminus of A tRNA+ The 39 end of P tRNA may be shielded from hydroxyl radicals by its interactions with the 2250 loop (Samaha et al+, 1995) and other features of 23S rRNA+ These studies have focused on two particular sets of tRNA binding complexes+ There are currently believed to be as many as eight (or possibly more) identifiable binding states for tRNA …”

Section: Discussionmentioning

confidence: 99%

Calculation of the relative geometry of tRNAs in the ribosome from directed hydroxyl-radical probing data

Joseph¹,

Whirl²,

Kondo³

et al. 2000

RNA

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The many interactions of tRNA with the ribosome are fundamental to protein synthesis. During the peptidyl transferase reaction, the acceptor ends of the aminoacyl and peptidyl tRNAs must be in close proximity to allow peptide bond formation, and their respective anticodons must base pair simultaneously with adjacent trinucleotide codons on the mRNA. The two tRNAs in this state can be arranged in two nonequivalent general configurations called the R and S orientations, many versions of which have been proposed for the geometry of tRNAs in the ribosome. Here, we report the combined use of computational analysis and tethered hydroxyl-radical probing to constrain their arrangement. We used Fe(II) tethered to the 59 end of anticodon stem-loop analogs (ASLs) of tRNA and to the 59 end of deacylated tRNA Phe to generate hydroxyl radicals that probe proximal positions in the backbone of adjacent tRNAs in the 70S ribosome. We inferred probe-target distances from the resulting RNA strand cleavage intensities and used these to calculate the mutual arrangement of A-site and P-site tRNAs in the ribosome, using three different structure estimation algorithms. The two tRNAs are constrained to the S configuration with an angle of about 458 between the respective planes of the molecules. The terminal phosphates of 39CCA are separated by 23 Å when using the tRNA crystal conformations, and the anticodon arms of the two tRNAs are sufficiently close to interact with adjacent codons in mRNA.

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“…Ribosomal Protein L10e Confers Sordarin Resistance-Identification of the large ribosomal subunit as the determinant of sordarin resistance, in addition to the biochemical and genetic associations of the stalk proteins with EF-G or eEF2 (11,13,(15)(16)(17)(18)(19), focused our efforts on the ribosomal stalk proteins. Because both fusidic acid and sordarin stabilize the eEF2-GDPribosome complex, we hypothesized that proteins in the stalk were candidate biochemical targets of sordarin.…”

Section: L10e Mutants and Eef2 Functionmentioning

confidence: 99%

“…Chemical cross-links have been observed between EF-Tu and EF-G and the L7/L12 complex (reviewed in Ref. 16). Of particular relevance to the present case, cross-links are observed between the EF-G and L7/ L12 proteins in the presence of the nonhydrolyzable GTP analog GMPPCP, but not in the presence of GDP and fusidic acid (17,18).…”

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confidence: 99%

Mutations in Ribosomal Protein L10e Confer Resistance to the Fungal-specific Eukaryotic Elongation Factor 2 Inhibitor Sordarin

Justice

Hsu

et al. 1999

Journal of Biological Chemistry

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The natural product sordarin, a tetracyclic diterpene glycoside, selectively inhibits fungal protein synthesis by impairing the function of eukaryotic elongation factor 2 (eEF2). Sordarin and its derivatives bind to the eEF2-ribosome-nucleotide complex in sensitive fungi, stabilizing the post-translocational GDP form. We have previously described a class of Saccharomyces cerevisiae mutants that exhibit resistance to varying levels of sordarin and have identified amino acid substitutions in yeast eEF2 that confer sordarin resistance. We now report on a second class of sordarin-resistant mutants. Biochemical and molecular genetic analysis of these mutants demonstrates that sordarin resistance is dependent on the essential large ribosomal subunit protein L10e in S. cerevisiae. Five unique L10e alleles were characterized and sequenced, and several nucleotide changes that differ from the wild-type sequence were identified. Changes that result in the resistance phenotype map to 4 amino acid substitutions and 1 amino acid deletion clustered in a conserved 10-amino acid region of L10e. Like the previously identified eEF2 mutations, the mutant ribosomes show reduced sordarin-conferred stabilization of the eEF2-nucleotide-ribosome complex. To our knowledge, this report provides the first description of ribosomal protein mutations affecting translocation. These results and our previous observations with eEF2 suggest a functional linkage between L10e and eEF2.Eukaryotic elongation factor 2 (eEF2) 1 and its prokaryotic counterpart, elongation factor G (EF-G), promote the translocation of the ribosome along messenger RNA during the elongation phase of protein synthesis. Hydrolysis of GTP to GDP drives translocation and is associated with a presumed conformational change in eEF2. Sordarin (1) and its analogs are fungal-specific translation inhibitors (2, 3) that bind to the eEF2-ribosome-GDP complex in Saccharomyces cerevisiae, stabilizing the post-translocational GDP form in a manner similar to that of fusidic acid (3). However, in contrast to fusidic acid, which binds both EF-G and eEF2 and is a general translocation inhibitor, sordarin inhibits translation only in susceptible fungi, deriving its unique specificity from the source of eEF2 (3-5). The observation that eEF2 is the major determinant of sordarin specificity was confirmed by the identification of 15 unique sordarin-resistant alleles of EFT1 and EFT2 that encode eEF2 in S. cerevisiae. In our original characterization of 21 sordarin-resistant mutants, five mutations were not linked to the EFT1 or EFT2 genes. In this work, we show that these five mutations map to the essential ribosomal protein L10e.The ribosome, although not contributing significantly to the fungal specificity of sordarin, is a critical partner in forming the stabilized post-translocational complex (3). Detection of a complex between fungal eEF2 and a labeled sordarin analog is strongly dependent upon the presence of ribosomes. L10, the prokaryotic counterpart of S. cerevisiae L10e, has been localiz...

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The Elongation Phase of Protein Synthesis

Cited by 61 publications

References 179 publications

Elongation Factor 2 as a Novel Target for Selective Inhibition of Fungal Protein Synthesis

Elongation Factor 2 as a Novel Target for Selective Inhibition of Fungal Protein Synthesis

Calculation of the relative geometry of tRNAs in the ribosome from directed hydroxyl-radical probing data

Mutations in Ribosomal Protein L10e Confer Resistance to the Fungal-specific Eukaryotic Elongation Factor 2 Inhibitor Sordarin

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