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Nilgun E. Tumer - One of the best experts on this subject based on the ideXlab platform.

  • Ribosome depurination by ricin leads to inhibition of endoplasmic reticulum stress induced hac1 mrna splicing on the Ribosome
    Journal of Biological Chemistry, 2019
    Co-Authors: Michael Pierce, Diana Vengsarkar, John E Mclaughlin, Jennifer Nielsen Kahn, Nilgun E. Tumer
    Abstract:

    Ricin undergoes retrograde transport to the endoplasmic reticulum (ER), and ricin toxin A chain (RTA) enters the cytosol from the ER. Previous reports indicated that RTA inhibits activation of the unfolded protein response (UPR) in yeast and in mammalian cells. Both precursor (preRTA) and mature form of RTA (mRTA) inhibited splicing of HAC1u (u for uninduced) mRNA, suggesting that UPR inhibition occurred on the cytosolic face of the ER. Here, we examined the role of Ribosome binding and depurination activity on inhibition of the UPR using mRTA mutants. An active-site mutant with very low depurination activity, which bound Ribosomes as WT RTA, did not inhibit HAC1u mRNA splicing. A Ribosome-binding mutant, which showed reduced binding to Ribosomes but retained depurination activity, inhibited HAC1u mRNA splicing. This mutant allowed separation of the UPR inhibition by RTA from cytotoxicity because it reduced the rate of depurination. The Ribosome-binding mutant inhibited the UPR without affecting IRE1 oligomerization or cleavage of HAC1u mRNA at the splice site junctions. Inhibition of the UPR correlated with the depurination level, suggesting that Ribosomes play a role in splicing of HAC1u mRNA. We show that HAC1u mRNA is associated with Ribosomes and does not get processed on depurinated Ribosomes, thereby inhibiting the UPR. These results demonstrate that RTA inhibits HAC1u mRNA splicing through its depurination activity on the Ribosome without directly affecting IRE1 oligomerization or the splicing reaction and provide evidence that IRE1 recognizes HAC1u mRNA that is associated with Ribosomes.

  • shiga toxin 1 is more dependent on the p proteins of the ribosomal stalk for depurination activity than shiga toxin 2
    The International Journal of Biochemistry & Cell Biology, 2011
    Co-Authors: Jiachi Chiou, Miguel Remacha, Juan P G Ballesta, Xiaoping Li, Nilgun E. Tumer
    Abstract:

    Abstract Shiga toxins produced by Escherichia coli O157:H7 are responsible for food poisoning and hemolytic uremic syndrome (HUS). The A subunits of Shiga toxins (Stx1A and Stx2A) inhibit translation by depurinating a specific adenine in the large rRNA. To determine if Stx1A and Stx2A require the ribosomal stalk for depurination, their activity and cytotoxicity were examined in the yeast P protein deletion mutants. Stx1A and Stx2A were less toxic and depurinated Ribosomes less in a strain lacking P1/P2 on the Ribosome and in the cytosol (ΔP2) than in a strain lacking P1/P2 on the Ribosome, but containing free P2 in the cytosol (ΔP1). To determine if cytoplasmic P proteins facilitated depurination, Stx1A and Stx2A were expressed in the P0ΔAB mutant, in which the binding sites for P1/P2 were deleted on the Ribosome, and P1/P2 accumulated in the cytosol. Stx1A was less toxic and depurinated Ribosomes less in P0ΔAB, suggesting that intact binding sites for P1/P2 were critical. In contrast, Stx2A was toxic and depurinated Ribosomes in P0ΔAB as in wild type, suggesting that it did not require the P1/P2 binding sites. Depurination of ΔP1, but not P0ΔAB Ribosomes increased upon addition of purified P1α/P2β in vitro, and the increase was greater for Stx1 than for Stx2. We conclude that cytoplasmic P proteins stimulate depurination by Stx1 by facilitating the access of the toxin to the Ribosome. Although ribosomal stalk is important for Stx1 and Stx2 to depurinate the Ribosome, Stx2 is less dependent on the stalk proteins for activity than Stx1 and can depurinate Ribosomes with an incomplete stalk better than Stx1.

  • the ribosomal stalk is required for Ribosome binding depurination of the rrna and cytotoxicity of ricin a chain in saccharomyces cerevisiae
    Molecular Microbiology, 2008
    Co-Authors: Jiachi Chiou, Miguel Remacha, Juan P G Ballesta, Xiaoping Li, Nilgun E. Tumer
    Abstract:

    : Ribosome inactivating proteins (RIPs) like ricin, pokeweed antiviral protein (PAP) and Shiga-like toxins 1 and 2 (Stx1 and Stx2) share the same substrate, the alpha-sarcin/ricin loop, but differ in their specificities towards prokaryotic and eukaryotic Ribosomes. Ricin depurinates the eukaryotic Ribosomes more efficiently than the prokaryotic Ribosomes, while PAP can depurinate both types of Ribosomes. Accumulating evidence suggests that different docking sites on the Ribosome might be used by different RIPs, providing a basis for understanding the mechanism underlying their kingdom specificity. Our previous results demonstrated that PAP binds to the ribosomal protein L3 to depurinate the alpha-sarcin/ricin loop and binding of PAP to L3 was critical for its cytotoxicity. Here, we used surface plasmon resonance to demonstrate that ricin toxin A chain (RTA) binds to the P1 and P2 proteins of the ribosomal stalk in Saccharomyces cerevisiae. Ribosomes from the P protein mutants were depurinated less than the wild-type Ribosomes when treated with RTA in vitro. Ribosome depurination was reduced when RTA was expressed in the DeltaP1 and DeltaP2 mutants in vivo and these mutants were more resistant to the cytotoxicity of RTA than the wild-type cells. We further show that while RTA, Stx1 and Stx2 have similar requirements for Ribosome depurination, PAP has different requirements, providing evidence that the interaction of RIPs with different ribosomal proteins is responsible for their Ribosome specificity.

  • generation of pokeweed antiviral protein mutations in saccharomyces cerevisiae evidence that Ribosome depurination is not sufficient for cytotoxicity
    Nucleic Acids Research, 2004
    Co-Authors: Katalin A. Hudak, Bijal A Parikh, Rong Di, Marianne Baricevic, Mirjana Seskar, Maria Santana, Nilgun E. Tumer
    Abstract:

    Pokeweed antiviral protein (PAP) is a Ribosome-inactivating protein that depurinates the highly conserved α-sarcin/ricin loop in the large rRNA. Here, using site-directed mutagenesis and systematic deletion analysis from the 5′ and the 3′ ends of the PAP cDNA, we identified the amino acids important for Ribosome depurination and cytotoxicity of PAP. Truncating the first 16 amino acids of PAP eliminated its cytotoxicity and the ability to depurinate Ribosomes. Ribosome depurination gradually decreased upon the sequential deletion of C-terminal amino acids and was abolished when a stop codon was introduced at Glu-244. Cytotoxicity of the C-terminal deletion mutants was lost before their ability to depurinate Ribosomes. Mutations in Tyr-123 at the active site affected cytotoxicity without altering the Ribosome depurination ability. Total translation was not inhibited in yeast expressing the non-toxic Tyr-123 mutants, although Ribosomes were depurinated. These mutants depurinated Ribosomes only during their translation and could not depurinate Ribosomes in trans in a translation-independent manner. A mutation in Leu-71 in the central domain affected cytotoxicity without altering the ability to depurinate Ribosomes in trans and inhibit translation. These results demonstrate that the ability to depurinate Ribosomes in trans in a catalytic manner is required for the inhibition of translation, but is not sufficient for cytotoxicity.

Michael C. Jewett - One of the best experts on this subject based on the ideXlab platform.

  • in vitro Ribosome synthesis and evolution through Ribosome display
    Nature Communications, 2020
    Co-Authors: Michael J Hammerling, Brian R Fritz, Danielle J Yoesep, Erik D Carlson, Michael C. Jewett
    Abstract:

    Directed evolution of the Ribosome for expanded substrate incorporation and novel functions is challenging because the requirement of cell viability limits the mutations that can be made. Here we address this challenge by combining cell-free synthesis and assembly of translationally competent Ribosomes with Ribosome display to develop a fully in vitro methodology for Ribosome synthesis and evolution (called RISE). We validate the RISE method by selecting active genotypes from a ~1.7 × 107 member library of ribosomal RNA (rRNA) variants, as well as identifying mutant Ribosomes resistant to the antibiotic clindamycin from a library of ~4 × 103 rRNA variants. We further demonstrate the prevalence of positive epistasis in resistant genotypes, highlighting the importance of such interactions in selecting for new function. We anticipate that RISE will facilitate understanding of molecular translation and enable selection of Ribosomes with altered properties. Directed evolution of the Ribosome is challenging because the requirement of cell viability limits the mutations that can be made. Here the authors develop a platform for in vitro Ribosome synthesis and evolution (RISE) to overcome these constraints.

  • in vitro Ribosome synthesis and evolution through Ribosome display
    bioRxiv, 2019
    Co-Authors: Michael J Hammerling, Brian R Fritz, Danielle J Yoesep, Erik D Carlson, Michael C. Jewett
    Abstract:

    Directed evolution of the Ribosome for expanded substrate incorporation and novel functions is challenging because the requirement of cell viability limits the mutations that can be made. However, our recent development of an integrated strategy for the in vitro synthesis and assembly of translationally competent Ribosomes (iSAT) enables the rapid generation of large libraries of Ribosome variants in a cell-free environment. Here we combine the iSAT system with Ribosome display to develop a fully in vitro methodology for Ribosome synthesis and evolution (called RISE). We validate this method by selecting highly active genotypes which are resistant to the antibiotic clindamycin from a library of Ribosome variants. We further demonstrate the prevalence of positive epistasis in successful genotypes, highlighting the importance of such interactions in selecting for new function. We anticipate that RISE will facilitate understanding of molecular translation and enable selection of Ribosomes with altered properties.

  • implications of macromolecular crowding and reducing conditions for in vitro Ribosome construction
    Nucleic Acids Research, 2015
    Co-Authors: Brian R Fritz, Osman K Jamil, Michael C. Jewett
    Abstract:

    In vitro construction of Escherichia coli Ribosomes could elucidate a deeper understanding of these complex molecular machines and make possible the production of synthetic variants with new functions. Toward this goal, we recently developed an integrated synthesis, assembly and translation (iSAT) system that allows for co-activation of ribosomal RNA (rRNA) transcription and Ribosome assembly, mRNA transcription and protein translation without intact cells. Here, we discovered that macromolecular crowding and reducing agents increase overall iSAT protein synthesis; the combination of 6% w/v Ficoll 400 and 2 mM DTBA yielded approximately a five-fold increase in overall iSAT protein synthesis activity. By utilizing a fluorescent RNA aptamer, fluorescent reporter proteins and Ribosome sedimentation analysis, we showed that crowding agents increase iSAT yields by enhancing translation while reducing agents increase rRNA transcription and Ribosome assembly. Finally, we showed that iSAT Ribosomes possess ∼70% of the protein synthesis activity of in vivo-assembled E. coli Ribosomes. This work improves iSAT protein synthesis through the addition of crowding and reducing agents, provides a thorough understanding of the effect of these additives within the iSAT system and demonstrates how iSAT allows for manipulation and analysis of Ribosome biogenesis in the context of an in vitro transcription-translation system.

  • in vitro integration of ribosomal rna synthesis Ribosome assembly and translation
    Molecular Systems Biology, 2013
    Co-Authors: Brian R Fritz, Michael C. Jewett, Laura E Timmerman, George M Church
    Abstract:

    Purely in vitro Ribosome synthesis could provide a critical step towards unraveling the systems biology of Ribosome biogenesis, constructing minimal cells from defined components, and engineering Ribosomes with new functions. Here, as an initial step towards this goal, we report a method for constructing Escherichia coli Ribosomes in crude S150 E. coli extracts. While conventional methods for E. coli Ribosome reconstitution are non-physiological, our approach attempts to mimic chemical conditions in the cytoplasm, thus permitting several biological processes to occur simultaneously. Specifically, our integrated synthesis, assembly, and translation (iSAT) technology enables one-step co-activation of rRNA transcription, assembly of transcribed rRNA with native ribosomal proteins into functional Ribosomes, and synthesis of active protein by these Ribosomes in the same compartment. We show that iSAT makes possible the in vitro construction of modified Ribosomes by introducing a 23S rRNA mutation that mediates resistance against clindamycin. We anticipate that iSAT will aid studies of Ribosome assembly and open new avenues for making Ribosomes with altered properties.

Mans Ehrenberg - One of the best experts on this subject based on the ideXlab platform.

  • complementary roles of initiation factor 1 and Ribosome recycling factor in 70s Ribosome splitting
    The EMBO Journal, 2008
    Co-Authors: Michael Y Pavlov, Ayman Antoun, Martin Lovmar, Mans Ehrenberg
    Abstract:

    We demonstrate that Ribosomes containing a messenger RNA (mRNA) with a strong Shine–Dalgarno sequence are rapidly split into subunits by initiation factors 1 (IF1) and 3 (IF3), but slowly split by Ribosome recycling factor (RRF) and elongation factor G (EF-G). Post-termination-like (PTL) Ribosomes containing mRNA and a P-site-bound deacylated transfer RNA (tRNA) are split very rapidly by RRF and EF-G, but extremely slowly by IF1 and IF3. Vacant Ribosomes are split by RRF/EF-G much more slowly than PTL Ribosomes and by IF1/IF3 much more slowly than mRNA-containing Ribosomes. These observations reveal complementary splitting of different ribosomal complexes by IF1/IF3 and RRF/EF-G, and suggest the existence of two major pathways for Ribosome splitting into subunits in the living cell. We show that the identity of the deacylated tRNA in the PTL Ribosome strongly affects the rate by which it is split by RRF/EF-G and that IF3 is involved in the mechanism of Ribosome splitting by IF1/IF3 but not by RRF/EF-G. With support from our experimental data, we discuss the principally different mechanisms of Ribosome splitting by IF1/IF3 and by RRF/EF-G.

  • Ribosome release factor RF4 and termination factor RF3 are involved in dissociation of peptidyl-tRNA from the Ribosome
    The EMBO Journal, 1998
    Co-Authors: Valérie Heurgué-hamard, Céline Leboeuf, Guido Grentzmann, Reza Karimi, Jane Macdougall, Liliana Mora, Mans Ehrenberg, Richard H. Buckingham
    Abstract:

    Peptidyl-tRNA dissociation from Ribosomes is an energetically costly but apparently inevitable process that accompanies normal protein synthesis. The drop-off products of these events are hydrolysed by peptidyl-tRNA hydrolase. Mutant selections have been made to identify genes involved in the drop-off of peptidyl-tRNA, using a thermosensitive peptidyl-tRNA hydrolase mutant in Escherichia coli. Transposon insertions upstream of the frr gene, which encodes RF4 (Ribosome release or recycling factor), restored growth to this mutant. The insertions impaired expression of the frr gene. Mutations inactivating prfC, encoding RF3 (release factor 3), displayed a similar phenotype. Conversely, production of RF4 from a plasmid increased the thermosensitivity of the peptidyl-tRNA hydrolase mutant. In vitro measurements of peptidyl-tRNA release from Ribosomes paused at stop signals or sense codons confirmed that RF3 and RF4 were able to stimulate peptidyl-tRNA release from Ribosomes, and showed that this action of RF4 required the presence of translocation factor EF2, known to be needed for the function of RF4 in Ribosome recycling. When present together, the three factors were able to stimulate release up to 12-fold. It is suggested that RF4 may displace peptidyl-tRNA from the Ribosome in a manner related to its proposed function in removing deacylated tRNA during Ribosome recycling.

Jamie H D Cate - One of the best experts on this subject based on the ideXlab platform.

  • defects in the assembly of Ribosomes selected for β amino acid incorporation
    Biochemistry, 2019
    Co-Authors: Fred R Ward, Zoe L Watson, Alanna Schepartz, Jamie H D Cate
    Abstract:

    Ribosome engineering has emerged as a promising field in synthetic biology, particularly concerning the production of new sequence-defined polymers. Mutant Ribosomes have been developed that improve the incorporation of several nonstandard monomers including d-amino acids, dipeptides, and β-amino acids into polypeptide chains. However, there remains little mechanistic understanding of how these Ribosomes catalyze incorporation of these new substrates. Here, we probed the properties of a mutant Ribosome-P7A7-evolved for better in vivo β-amino acid incorporation through in vitro biochemistry and cryo-electron microscopy. Although P7A7 is a functional Ribosome in vivo, it is inactive in vitro, and assembles poorly into 70S Ribosome complexes. Structural characterization revealed large regions of disorder in the peptidyltransferase center and nearby features, suggesting a defect in assembly. Comparison of RNA helix and ribosomal protein occupancy with other assembly intermediates revealed that P7A7 is stalled at a late stage in Ribosome assembly, explaining its weak activity. These results highlight the importance of ensuring efficient Ribosome assembly during Ribosome engineering toward new catalytic abilities.

  • defects in the assembly of Ribosomes selected for β amino acid incorporation
    bioRxiv, 2019
    Co-Authors: Fred R Ward, Zoe L Watson, Alanna Schepartz, Jamie H D Cate
    Abstract:

    Ribosome engineering has emerged as a promising field in synthetic biology, particularly concerning the production of new sequence-defined polymers. Mutant Ribosomes have been developed that improve the incorporation of several non-standard monomers including D-amino acids, dipeptides, and β-amino acids into polypeptide chains. However, there remains little mechanistic understanding of how these Ribosomes catalyze incorporation of these new substrates. Here we probed the properties of a mutant Ribosome-P7A7-evolved for better in vivo β-amino acid incorporation through in vitro; biochemistry and cryo-electron microscopy. Although P7A7 is a functional Ribosome in vivo, it is inactive in vitro,and assembles poorly into 70S complexes. Structural characterization revealed large regions of disorder in the peptidyl transferase center and nearby features, suggesting a defect in assembly. Comparison of RNA helix and ribosomal protein occupancy with other assembly intermediates revealed that P7A7 is stalled at a late stage in Ribosome assembly, explaining its weak activity. These results highlight the importance of ensuring efficient Ribosome assembly during Ribosome engineering towards new catalytic abilities.

Meengan Frances Yap - One of the best experts on this subject based on the ideXlab platform.

  • the hibernating 100s complex is a target of Ribosome recycling factor and elongation factor g in staphylococcus aureus
    Journal of Biological Chemistry, 2020
    Co-Authors: Arnab Basu, Kathryn E Shields, Meengan Frances Yap
    Abstract:

    The formation of translationally inactive 70S dimers (called 100S Ribosomes) by hibernation-promoting factor is a widespread survival strategy among bacteria. Ribosome dimerization is thought to be reversible, with the dissociation of the 100S complexes enabling Ribosome recycling for participation in new rounds of translation. The precise pathway of 100S Ribosome recycling has been unclear. We previously found that the heat-shock GTPase HflX in the human pathogen Staphylococcus aureus is a minor disassembly factor. Cells lacking hflX do not accumulate 100S Ribosomes unless they are subjected to heat exposure, suggesting the existence of an alternative pathway during nonstressed conditions. Here, we provide biochemical and genetic evidence that two essential translation factors, Ribosome-recycling factor (RRF) and GTPase elongation factor G (EF-G), synergistically split 100S Ribosomes in a GTP-dependent but tRNA translocation-independent manner. We found that although HflX and the RRF/EF-G pair are functionally interchangeable, HflX is expressed at low levels and is dispensable under normal growth conditions. The bacterial RRF/EF-G pair was previously known to target only the post-termination 70S complexes; our results reveal a new role in the reversal of Ribosome hibernation that is intimately linked to bacterial pathogenesis, persister formation, stress responses, and Ribosome integrity.