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Terri Goss Kinzy - One of the best experts on this subject based on the ideXlab platform.
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methylation of Translation Elongation factor 1a by the mettl10 like see1 methyltransferase facilitates tombusvirus replication in yeast and plants
Virology, 2014Co-Authors: Zhenghe Li, Zsuzsanna Sasvari, Paulina Alatriste Gonzalez, Terri Goss Kinzy, Peter D NagyAbstract:Abstract Replication of tombusviruses and other plus-strand RNA viruses depends on several host factors that are recruited into viral replicase complexes. Previous studies have shown that eukaryotic Translation Elongation factor 1A (eEF1A) is one of the resident host proteins in the highly purified tombusvirus replicase complex. In this paper, we show that methylation of eEF1A by the METTL10-like See1p methyltransferase is required for tombusvirus and unrelated nodavirus RNA replication in yeast model host. Similar to the effect of SEE1 deletion, yeast expressing only a mutant form of eEF1A lacking the 4 known lysines subjected to methylation supported reduced TBSV accumulation. We show that the half-life of several viral replication proteins is decreased in see1Δ yeast or when a mutated eEF1A was expressed as a sole source for eEF1A. Silencing of the plant ortholog of See1 methyltransferase also decreased tombusvirus RNA accumulation in Nicotiana benthamiana.
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adp ribosylation of Translation Elongation factor 2 by diphtheria toxin in yeast inhibits Translation and cell separation
Journal of Biological Chemistry, 2013Co-Authors: Maria K Mateyak, Terri Goss KinzyAbstract:Eukaryotic Translation Elongation factor 2 (eEF2) facilitates the movement of the peptidyl tRNA-mRNA complex from the A site of the ribosome to the P site during protein synthesis. ADP-ribosylation (ADPR) of eEF2 by bacterial toxins on a unique diphthamide residue inhibits its translocation activity, but the mechanism is unclear. We have employed a hormone-inducible diphtheria toxin (DT) expression system in Saccharomyces cerevisiae which allows for the rapid induction of ADPR-eEF2 to examine the effects of DT in vivo. ADPR of eEF2 resulted in a decrease in total protein synthesis consistent with a defect in Translation Elongation. Association of eEF2 with polyribosomes, however, was unchanged upon expression of DT. Upon prolonged exposure to DT, cells with an abnormal morphology and increased DNA content accumulated. This observation was specific to DT expression and was not observed when Translation Elongation was inhibited by other methods. Examination of these cells by electron microscopy indicated a defect in cell separation following mitosis. These results suggest that expression of proteins late in the cell cycle is particularly sensitive to inhibition by ADPR-eEF2. Background: Diphtheria toxin inhibits Translation by ADP-ribosylation of Elongation factor 2. Results: Diphtheria toxin expression results in accumulation of cells that fail to separate following mitosis. Conclusion: Diphtheria toxin expression has unique effects on translocation and the production of specific proteins. Significance: Understanding how ADP-ribosylated Elongation factor 2 affects translocation will increase knowledge of Translation and potentially lead to new treatments for toxin exposure.
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synergistic roles of eukaryotic Translation Elongation factors 1bγ and 1a in stimulation of tombusvirus minus strand synthesis
PLOS Pathogens, 2011Co-Authors: Zsuzsanna Sasvari, Lara S Izotova, Terri Goss Kinzy, Peter D NagyAbstract:Host factors are recruited into viral replicase complexes to aid replication of plus-strand RNA viruses. In this paper, we show that deletion of eukaryotic Translation Elongation factor 1Bgamma (eEF1Bγ) reduces Tomato bushy stunt virus (TBSV) replication in yeast host. Also, knock down of eEF1Bγ level in plant host decreases TBSV accumulation. eEF1Bγ binds to the viral RNA and is one of the resident host proteins in the tombusvirus replicase complex. Additional in vitro assays with whole cell extracts prepared from yeast strains lacking eEF1Bγ demonstrated its role in minus-strand synthesis by opening of the structured 3′ end of the viral RNA and reducing the possibility of re-utilization of (+)-strand templates for repeated (-)-strand synthesis within the replicase. We also show that eEF1Bγ plays a synergistic role with eukaryotic Translation Elongation factor 1A in tombusvirus replication, possibly via stimulation of the proper positioning of the viral RNA-dependent RNA polymerase over the promoter region in the viral RNA template.These roles for Translation factors during TBSV replication are separate from their canonical roles in host and viral protein Translation.
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Translation Elongation factor 1a facilitates the assembly of the tombusvirus replicase and stimulates minus strand synthesis
PLOS Pathogens, 2010Co-Authors: Zhenghe Li, Steven Tupman, Anthony M Esposito, Terri Goss Kinzy, Judit Pogany, Peter D NagyAbstract:Replication of plus-strand RNA viruses depends on host factors that are recruited into viral replicase complexes. Previous studies showed that eukaryotic Translation Elongation factor (eEF1A) is one of the resident host proteins in the highly purified tombusvirus replicase complex. Using a random library of eEF1A mutants, we identified one mutant that decreased and three mutants that increased Tomato bushy stunt virus (TBSV) replication in a yeast model host. Additional in vitro assays with whole cell extracts prepared from yeast strains expressing the eEF1A mutants demonstrated several functions for eEF1A in TBSV replication: facilitating the recruitment of the viral RNA template into the replicase complex; the assembly of the viral replicase complex; and enhancement of the minus-strand synthesis by promoting the initiation step. These roles for eEF1A are separate from its canonical role in host and viral protein Translation, emphasizing critical functions for this abundant cellular protein during TBSV replication.
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Translation Elongation factor 1a facilitates the assembly of the tombusvirus replicase and stimulates minus strand synthesis
PLOS Pathogens, 2010Co-Authors: Zhenghe Li, Steven Tupman, Anthony M Esposito, Terri Goss Kinzy, Judit Pogany, Peter D NagyAbstract:Replication of plus-strand RNA viruses depends on host factors that are recruited into viral replicase complexes. Previous studies showed that eukaryotic Translation Elongation factor (eEF1A) is one of the resident host proteins in the highly purified tombusvirus replicase complex. Using a random library of eEF1A mutants, we identified one mutant that decreased and three mutants that increased Tomato bushy stunt virus (TBSV) replication in a yeast model host. Additional in vitro assays with whole cell extracts prepared from yeast strains expressing the eEF1A mutants demonstrated several functions for eEF1A in TBSV replication: facilitating the recruitment of the viral RNA template into the replicase complex; the assembly of the viral replicase complex; and enhancement of the minus-strand synthesis by promoting the initiation step. These roles for eEF1A are separate from its canonical role in host and viral protein Translation, emphasizing critical functions for this abundant cellular protein during TBSV replication.
Daniel N. Wilson - One of the best experts on this subject based on the ideXlab platform.
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yeast Translation Elongation factor eef3 promotes late stages of trna translocation
The EMBO Journal, 2021Co-Authors: Namit Ranjan, Rachel Green, Daniel N. Wilson, Agnieszka A Pochopien, Bertrand Beckert, Sandra Blanchet, Marina V RodninaAbstract:In addition to the conserved Translation Elongation factors eEF1A and eEF2, fungi require a third essential Elongation factor, eEF3. While eEF3 has been implicated in tRNA binding and release at the ribosomal A and E sites, its exact mechanism of action is unclear. Here, we show that eEF3 acts at the mRNA-tRNA translocation step by promoting the dissociation of the tRNA from the E site, but independent of aminoacyl-tRNA recruitment to the A site. Depletion of eEF3 in vivo leads to a general slowdown in Translation Elongation due to accumulation of ribosomes with an occupied A site. Cryo-EM analysis of native eEF3-ribosome complexes shows that eEF3 facilitates late steps of translocation by favoring non-rotated ribosomal states, as well as by opening the L1 stalk to release the E-site tRNA. Additionally, our analysis provides structural insights into novel Translation Elongation states, enabling presentation of a revised yeast Translation Elongation cycle.
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stall no more at polyproline stretches with the Translation Elongation factors ef p and if 5a
Molecular Microbiology, 2016Co-Authors: Kirsten Jung, Jürgen Lassak, Daniel N. WilsonAbstract:Synthesis of polyproline proteins leads to Translation arrest. To overcome this ribosome stalling effect, bacteria depend on a specialized Translation Elongation factor P (EF-P), being orthologous and functionally identical to eukaryotic/archaeal Elongation factor e/aIF-5A (recently renamed 'EF5'). EF-P binds to the stalled ribosome between the peptidyl-tRNA binding and tRNA-exiting sites, and stimulates peptidyl-transferase activity, thus allowing Translation to resume. In their active form, both EF-P and e/aIF-5A are post-Translationally modified at a positively charged residue, which protrudes toward the peptidyl-transferase center when bound to the ribosome. While archaeal and eukaryotic IF-5A strictly depend on (deoxy-) hypusination (hypusinylation) of a conserved lysine, bacteria have evolved diverse analogous modification strategies to activate EF-P. In Escherichia coli and Salmonella enterica a lysine is extended by β-lysinylation and subsequently hydroxylated, whereas in Pseudomonas aeruginosa and Shewanella oneidensis an arginine in the equivalent position is rhamnosylated. Inactivation of EF-P, or the corresponding modification systems, reduces not only bacterial fitness, but also impairs virulence. Here, we review the function of EF-P and IF-5A and their unusual postTranslational protein modifications.
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arginine rhamnosylation as new strategy to activate Translation Elongation factor p
Nature Chemical Biology, 2015Co-Authors: Jürgen Lassak, Agata L. Starosta, Eva C Keilhauer, Maximilian J L J Furst, Kristin Wuichet, Julia Godeke, Jhongmin Chen, Lotte Sogaardandersen, Jurgen Rohr, Daniel N. WilsonAbstract:Ribosome stalling at polyproline stretches is common and fundamental. In bacteria, Translation Elongation factor P (EF-P) rescues such stalled ribosomes, but only when it is post-Translationally activated. In Escherichia coli, activation of EF-P is achieved by (R)-β-lysinylation and hydroxylation of a conserved lysine. Here we have unveiled a markedly different modification strategy in which a conserved arginine of EF-P is rhamnosylated by a glycosyltransferase (EarP) using dTDP-L-rhamnose as a substrate. This is to our knowledge the first report of N-linked protein glycosylation on arginine in bacteria and the first example in which a glycosylated side chain of a Translation Elongation factor is essential for function. Arginine-rhamnosylation of EF-P also occurs in clinically relevant bacteria such as Pseudomonas aeruginosa. We demonstrate that the modification is needed to develop pathogenicity, making EarP and dTDP-L-rhamnose-biosynthesizing enzymes ideal targets for antibiotic development.
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Translation Elongation Factor EF-P Alleviates Ribosome Stalling at Polyproline Stretches
Science (New York N.Y.), 2012Co-Authors: Susanne Ude, Jürgen Lassak, Agata L. Starosta, Tobias Kraxenberger, Daniel N. Wilson, Kirsten JungAbstract:Translation Elongation factor P (EF-P) is critical for virulence in bacteria. EF-P is present in all bacteria and orthologous to archaeal and eukaryotic initiation factor 5A, yet the biological function has so far remained enigmatic. Here, we demonstrate that EF-P is an Elongation factor that enhances Translation of polyproline-containing proteins: In the absence of EF-P, ribosomes stall at polyproline stretches, whereas the presence of EF-P alleviates the Translational stalling. Moreover, we demonstrate the physiological relevance of EF-P to fine-tune the expression of the polyproline-containing pH receptor CadC to levels necessary for an appropriate stress response. Bacterial, archaeal, and eukaryotic cells have hundreds to thousands of polyproline-containing proteins of diverse function, suggesting that EF-P and a/eIF-5A are critical for copy-number adjustment of multiple pathways across all kingdoms of life.
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lys34 of Translation Elongation factor ef p is hydroxylated by yfcm
Nature Chemical Biology, 2012Co-Authors: Lauri Peil, Agata L. Starosta, Kai Virumae, Gemma C Atkinson, Tanel Tenson, Jaanus Remme, Daniel N. WilsonAbstract:Lys34 of the conserved Translation Elongation factor P (EF-P) is post-Translationally lysinylated by YjeK and YjeA--a modification that is critical for bacterial virulence. Here we show that the currently accepted Escherichia coli EF-P modification pathway is incomplete and lacks a final hydroxylation step mediated by YfcM, an enzyme distinct from deoxyhypusine hydroxylase that catalyzes the final maturation step of eukaryotic initiation factor 5A, the eukaryotic EF-P homolog.
Peter D Nagy - One of the best experts on this subject based on the ideXlab platform.
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methylation of Translation Elongation factor 1a by the mettl10 like see1 methyltransferase facilitates tombusvirus replication in yeast and plants
Virology, 2014Co-Authors: Zhenghe Li, Zsuzsanna Sasvari, Paulina Alatriste Gonzalez, Terri Goss Kinzy, Peter D NagyAbstract:Abstract Replication of tombusviruses and other plus-strand RNA viruses depends on several host factors that are recruited into viral replicase complexes. Previous studies have shown that eukaryotic Translation Elongation factor 1A (eEF1A) is one of the resident host proteins in the highly purified tombusvirus replicase complex. In this paper, we show that methylation of eEF1A by the METTL10-like See1p methyltransferase is required for tombusvirus and unrelated nodavirus RNA replication in yeast model host. Similar to the effect of SEE1 deletion, yeast expressing only a mutant form of eEF1A lacking the 4 known lysines subjected to methylation supported reduced TBSV accumulation. We show that the half-life of several viral replication proteins is decreased in see1Δ yeast or when a mutated eEF1A was expressed as a sole source for eEF1A. Silencing of the plant ortholog of See1 methyltransferase also decreased tombusvirus RNA accumulation in Nicotiana benthamiana.
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synergistic roles of eukaryotic Translation Elongation factors 1bγ and 1a in stimulation of tombusvirus minus strand synthesis
PLOS Pathogens, 2011Co-Authors: Zsuzsanna Sasvari, Lara S Izotova, Terri Goss Kinzy, Peter D NagyAbstract:Host factors are recruited into viral replicase complexes to aid replication of plus-strand RNA viruses. In this paper, we show that deletion of eukaryotic Translation Elongation factor 1Bgamma (eEF1Bγ) reduces Tomato bushy stunt virus (TBSV) replication in yeast host. Also, knock down of eEF1Bγ level in plant host decreases TBSV accumulation. eEF1Bγ binds to the viral RNA and is one of the resident host proteins in the tombusvirus replicase complex. Additional in vitro assays with whole cell extracts prepared from yeast strains lacking eEF1Bγ demonstrated its role in minus-strand synthesis by opening of the structured 3′ end of the viral RNA and reducing the possibility of re-utilization of (+)-strand templates for repeated (-)-strand synthesis within the replicase. We also show that eEF1Bγ plays a synergistic role with eukaryotic Translation Elongation factor 1A in tombusvirus replication, possibly via stimulation of the proper positioning of the viral RNA-dependent RNA polymerase over the promoter region in the viral RNA template.These roles for Translation factors during TBSV replication are separate from their canonical roles in host and viral protein Translation.
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Translation Elongation factor 1a facilitates the assembly of the tombusvirus replicase and stimulates minus strand synthesis
PLOS Pathogens, 2010Co-Authors: Zhenghe Li, Steven Tupman, Anthony M Esposito, Terri Goss Kinzy, Judit Pogany, Peter D NagyAbstract:Replication of plus-strand RNA viruses depends on host factors that are recruited into viral replicase complexes. Previous studies showed that eukaryotic Translation Elongation factor (eEF1A) is one of the resident host proteins in the highly purified tombusvirus replicase complex. Using a random library of eEF1A mutants, we identified one mutant that decreased and three mutants that increased Tomato bushy stunt virus (TBSV) replication in a yeast model host. Additional in vitro assays with whole cell extracts prepared from yeast strains expressing the eEF1A mutants demonstrated several functions for eEF1A in TBSV replication: facilitating the recruitment of the viral RNA template into the replicase complex; the assembly of the viral replicase complex; and enhancement of the minus-strand synthesis by promoting the initiation step. These roles for eEF1A are separate from its canonical role in host and viral protein Translation, emphasizing critical functions for this abundant cellular protein during TBSV replication.
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Translation Elongation factor 1a facilitates the assembly of the tombusvirus replicase and stimulates minus strand synthesis
PLOS Pathogens, 2010Co-Authors: Zhenghe Li, Steven Tupman, Anthony M Esposito, Terri Goss Kinzy, Judit Pogany, Peter D NagyAbstract:Replication of plus-strand RNA viruses depends on host factors that are recruited into viral replicase complexes. Previous studies showed that eukaryotic Translation Elongation factor (eEF1A) is one of the resident host proteins in the highly purified tombusvirus replicase complex. Using a random library of eEF1A mutants, we identified one mutant that decreased and three mutants that increased Tomato bushy stunt virus (TBSV) replication in a yeast model host. Additional in vitro assays with whole cell extracts prepared from yeast strains expressing the eEF1A mutants demonstrated several functions for eEF1A in TBSV replication: facilitating the recruitment of the viral RNA template into the replicase complex; the assembly of the viral replicase complex; and enhancement of the minus-strand synthesis by promoting the initiation step. These roles for eEF1A are separate from its canonical role in host and viral protein Translation, emphasizing critical functions for this abundant cellular protein during TBSV replication.
Kirsten Jung - One of the best experts on this subject based on the ideXlab platform.
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structural basis for earp mediated arginine glycosylation of Translation Elongation factor ef p
Mbio, 2017Co-Authors: Ralph Krafczyk, Jurgen Rohr, Jakub Macošek, Daniel Gast, Swetlana Wunder, Pravin Kumar Ankush Jagtap, Prithiba Mitra, Amit Kumar Jha, Anja Hoffmannroder, Kirsten JungAbstract:Glycosylation is a universal strategy to postTranslationally modify proteins. The recently discovered arginine rhamnosylation activates the polyproline-specific bacterial Translation Elongation factor EF-P. EF-P is rhamnosylated on arginine 32 by the glycosyltransferase EarP. However, the enzymatic mechanism remains elusive. In the present study, we solved the crystal structure of EarP from Pseudomonas putida. The enzyme is composed of two opposing domains with Rossmann folds, thus constituting a B pattern-type glycosyltransferase (GT-B). While dTDP-beta-L-rhamnose is located within a highly conserved pocket of the C-domain, EarP recognizes the KOW-like N-domain of EF-P. Based on our data, we propose a structural model for arginine glycosylation by EarP. As EarP is essential for pathogenicity in P. aeruginosa, our study provides the basis for targeted inhibitor design. IMPORTANCE The structural and biochemical characterization of the EF-P-specific rhamnosyltransferase EarP not only provides the first molecular insights into arginine glycosylation but also lays the basis for targeted-inhibitor design against Pseudomonas aeruginosa infection.
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Structural basis for EarP-mediated arginine glycosylation of Translation Elongation factor EF-P
2017Co-Authors: Ralph Krafczyk, Jakub Macošek, Daniel Gast, Swetlana Wunder, Pravin Kumar Ankush Jagtap, Prithiba Mitra, Amit Kumar Jha, Juergen Rohr, Anja Hoffmann-röder, Kirsten JungAbstract:Glycosylation is a universal strategy to post-Translationally modify proteins. The recently discovered arginine rhamnosylation activates the polyproline specific bacterial Translation Elongation factor EF-P. EF-P is rhamnosylated on arginine 32 by the glycosyltransferase EarP. However, the enzymatic mechanism remains elusive. In the present study, we solved the crystal structure of EarP from Pseudomonas putida. The enzyme is composed of two opposing domains with Rossmann-folds, thus constituting a GT-B glycosyltransferase. While TDP-rhamnose is located within a highly conserved pocket of the C-domain, EarP recognizes the EF-P via its KOW-like N-domain. Based on our structural data combined with an in vitro / in vivo enzyme characterization, we propose a mechanism of inverting arginine glycosylation. As EarP is essential for pathogenicity in P. aeruginosa our study provides the basis for targeted inhibitor design.
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stall no more at polyproline stretches with the Translation Elongation factors ef p and if 5a
Molecular Microbiology, 2016Co-Authors: Kirsten Jung, Jürgen Lassak, Daniel N. WilsonAbstract:Synthesis of polyproline proteins leads to Translation arrest. To overcome this ribosome stalling effect, bacteria depend on a specialized Translation Elongation factor P (EF-P), being orthologous and functionally identical to eukaryotic/archaeal Elongation factor e/aIF-5A (recently renamed 'EF5'). EF-P binds to the stalled ribosome between the peptidyl-tRNA binding and tRNA-exiting sites, and stimulates peptidyl-transferase activity, thus allowing Translation to resume. In their active form, both EF-P and e/aIF-5A are post-Translationally modified at a positively charged residue, which protrudes toward the peptidyl-transferase center when bound to the ribosome. While archaeal and eukaryotic IF-5A strictly depend on (deoxy-) hypusination (hypusinylation) of a conserved lysine, bacteria have evolved diverse analogous modification strategies to activate EF-P. In Escherichia coli and Salmonella enterica a lysine is extended by β-lysinylation and subsequently hydroxylated, whereas in Pseudomonas aeruginosa and Shewanella oneidensis an arginine in the equivalent position is rhamnosylated. Inactivation of EF-P, or the corresponding modification systems, reduces not only bacterial fitness, but also impairs virulence. Here, we review the function of EF-P and IF-5A and their unusual postTranslational protein modifications.
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Translation Elongation Factor EF-P Alleviates Ribosome Stalling at Polyproline Stretches
Science (New York N.Y.), 2012Co-Authors: Susanne Ude, Jürgen Lassak, Agata L. Starosta, Tobias Kraxenberger, Daniel N. Wilson, Kirsten JungAbstract:Translation Elongation factor P (EF-P) is critical for virulence in bacteria. EF-P is present in all bacteria and orthologous to archaeal and eukaryotic initiation factor 5A, yet the biological function has so far remained enigmatic. Here, we demonstrate that EF-P is an Elongation factor that enhances Translation of polyproline-containing proteins: In the absence of EF-P, ribosomes stall at polyproline stretches, whereas the presence of EF-P alleviates the Translational stalling. Moreover, we demonstrate the physiological relevance of EF-P to fine-tune the expression of the polyproline-containing pH receptor CadC to levels necessary for an appropriate stress response. Bacterial, archaeal, and eukaryotic cells have hundreds to thousands of polyproline-containing proteins of diverse function, suggesting that EF-P and a/eIF-5A are critical for copy-number adjustment of multiple pathways across all kingdoms of life.
Rachel Green - One of the best experts on this subject based on the ideXlab platform.
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yeast Translation Elongation factor eef3 promotes late stages of trna translocation
The EMBO Journal, 2021Co-Authors: Namit Ranjan, Rachel Green, Daniel N. Wilson, Agnieszka A Pochopien, Bertrand Beckert, Sandra Blanchet, Marina V RodninaAbstract:In addition to the conserved Translation Elongation factors eEF1A and eEF2, fungi require a third essential Elongation factor, eEF3. While eEF3 has been implicated in tRNA binding and release at the ribosomal A and E sites, its exact mechanism of action is unclear. Here, we show that eEF3 acts at the mRNA-tRNA translocation step by promoting the dissociation of the tRNA from the E site, but independent of aminoacyl-tRNA recruitment to the A site. Depletion of eEF3 in vivo leads to a general slowdown in Translation Elongation due to accumulation of ribosomes with an occupied A site. Cryo-EM analysis of native eEF3-ribosome complexes shows that eEF3 facilitates late steps of translocation by favoring non-rotated ribosomal states, as well as by opening the L1 stalk to release the E-site tRNA. Additionally, our analysis provides structural insights into novel Translation Elongation states, enabling presentation of a revised yeast Translation Elongation cycle.
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Translation Elongation and recoding in eukaryotes
Cold Spring Harbor Perspectives in Biology, 2018Co-Authors: Thomas E Dever, Jonathan D Dinman, Rachel GreenAbstract:In this review, we highlight the current understanding of Translation Elongation and recoding in eukaryotes. In addition to providing an overview of the process, recent advances in our understanding of the role of the factor eIF5A in both Translation Elongation and termination are discussed. We also highlight mechanisms of Translation recoding with a focus on ribosomal frameshifting during Elongation. We see that the balance between the basic steps in Elongation and the less common recoding events is determined by the kinetics of the different processes as well as by specific sequence determinants.
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eif5a functions globally in Translation Elongation and termination
Molecular Cell, 2017Co-Authors: Anthony P Schuller, Thomas E Dever, Allen R Buskirk, Rachel GreenAbstract:Summary The eukaryotic Translation factor eIF5A, originally identified as an initiation factor, was later shown to promote Translation Elongation of iterated proline sequences. Using a combination of ribosome profiling and in vitro biochemistry, we report a much broader role for eIF5A in Elongation and uncover a critical function for eIF5A in termination. Ribosome profiling of an eIF5A-depleted strain reveals a global Elongation defect, with abundant ribosomes stalling at many sequences, not limited to proline stretches. Our data also show ribosome accumulation at stop codons and in the 3′ UTR, suggesting a global defect in termination in the absence of eIF5A. Using an in vitro reconstituted Translation system, we find that eIF5A strongly promotes the Translation of the stalling sequences identified by profiling and increases the rate of peptidyl-tRNA hydrolysis more than 17-fold. We conclude that eIF5A functions broadly in Elongation and termination, rationalizing its high cellular abundance and essential nature.
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inhibition of eukaryotic Translation Elongation by cycloheximide and lactimidomycin
Nature Chemical Biology, 2010Co-Authors: Tilman Schneiderpoetsch, Daniel E Eyler, Yongjun Dang, Shridhar Bhat, William C Merrick, Rachel Green, Ben Shen, Jun O LiuAbstract:Although the protein synthesis inhibitor cycloheximide (CHX) has been known for decades, its precise mechanism of action remains incompletely understood. The glutarimide portion of CHX is seen in a family of structurally related natural products including migrastatin, isomigrastatin and lactimidomycin (LTM). We found that LTM, isomigrastatin and analogs have a potent antiproliferative effect on tumor cell lines and selectively inhibit Translation. A systematic comparative study of the effects of CHX and LTM on protein synthesis revealed both similarities and differences between the two inhibitors. Both LTM and CHX were found to block the translocation step in Elongation. Footprinting experiments revealed protection of a single cytidine nucleotide (C3993) in the E-site of the 60S ribosomal subunit, thus defining a common binding pocket for the two inhibitors in the ribosome. These results shed new light on the molecular mechanism of inhibition of Translation Elongation by both CHX and LTM.
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hypusine containing protein eif5a promotes Translation Elongation
Nature, 2009Co-Authors: Preeti Saini, Daniel E Eyler, Rachel Green, Thomas E DeverAbstract:Various factors associate with the ribosome to assist in initiation, Elongation and termination. Textbook accounts of protein synthesis describe just two universally conserved Translation Elongation factors — EF-Tu/eEF1A and EF-G/eEF2. Now a study of protein synthesis in the yeast Saccharomyces cerevisiae repositions a factor previously thought to be associated with the initiation process, eIF5A, as a central player in Elongation. eIF5A is unusual in that it contains a rare amino acid, hypusine, that is required for its ability to stimulate Elongation. Based on the defects observed in the absence of eIF5A, it is proposed that the factor may function with eEF2 in the translocation step. Various factors associate with the ribosome to assist in initiation, Elongation and termination of Translation. Only two universal factors for Elongation have previously been identified; here, a factor previously thought to be associated with the initiation process, eIF5A, and which contains a rare amino acid, hypusine, is found to have a central role in Elongation. Translation Elongation factors facilitate protein synthesis by the ribosome. Previous studies identified two universally conserved Translation Elongation factors, EF-Tu in bacteria (known as eEF1A in eukaryotes) and EF-G (eEF2), which deliver aminoacyl-tRNAs to the ribosome and promote ribosomal translocation, respectively1. The factor eIF5A (encoded by HYP2 and ANB1 in Saccharomyces cerevisiae), the sole protein in eukaryotes and archaea to contain the unusual amino acid hypusine (Ne-(4-amino-2-hydroxybutyl)lysine)2, was originally identified based on its ability to stimulate the yield (endpoint) of methionyl-puromycin synthesis—a model assay for first peptide bond synthesis thought to report on certain aspects of Translation initiation3,4. Hypusine is required for eIF5A to associate with ribosomes5,6 and to stimulate methionyl-puromycin synthesis7. Because eIF5A did not stimulate earlier steps of Translation initiation8, and depletion of eIF5A in yeast only modestly impaired protein synthesis9, it was proposed that eIF5A function was limited to stimulating synthesis of the first peptide bond or that eIF5A functioned on only a subset of cellular messenger RNAs. However, the precise cellular role of eIF5A is unknown, and the protein has also been linked to mRNA decay, including the nonsense-mediated mRNA decay pathway10,11, and to nucleocytoplasmic transport12,13. Here we use molecular genetic and biochemical studies to show that eIF5A promotes Translation Elongation. Depletion or inactivation of eIF5A in the yeast S. cerevisiae resulted in the accumulation of polysomes and an increase in ribosomal transit times. Addition of recombinant eIF5A from yeast, but not a derivative lacking hypusine, enhanced the rate of tripeptide synthesis in vitro. Moreover, inactivation of eIF5A mimicked the effects of the eEF2 inhibitor sordarin, indicating that eIF5A might function together with eEF2 to promote ribosomal translocation. Because eIF5A is a structural homologue of the bacterial protein EF-P14,15, we propose that eIF5A/EF-P is a universally conserved Translation Elongation factor.
