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Thomas A Steitz - One of the best experts on this subject based on the ideXlab platform.
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Structures of Triacetyloleandomycin and Mycalamide A Bind to the Large Ribosomal Subunit of Haloarcula marismortui
Antimicrobial Agents and Chemotherapy, 2009Co-Authors: G. Gurel, Thomas A Steitz, Gregor Blaha, Peter B. MooreAbstract:Structures have been obtained for the complexes that triacetyloleandomycin and mycalamide A form with the Large Ribosomal Subunit of Haloarcula marismortui. Triacetyloleandomycin binds in the nascent peptide tunnel and inhibits the activity of ribosomes by blocking the growth of the nascent peptide chain. Mycalamide A binds to the E site and inhibits protein synthesis by occupying the space normally occupied by the CCA end of E-site-bound tRNAs.
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u2504 determines the species specificity of the a site cleft antibiotics the structures of tiamulin homoharringtonine and bruceantin bound to the ribosome
Journal of Molecular Biology, 2009Co-Authors: Guliz Gurel, Peter B. Moore, Thomas A Steitz, Gregor BlahaAbstract:Structures have been obtained for the complexes that tiamulin, homoharringtonine, and bruceantin form with the Large Ribosomal Subunit of Haloarcula marismortui at resolutions ranging from 2.65 to 3.2 A. They show that all these inhibitors block protein synthesis by competing with the amino acid side chains of incoming aminoacyl-tRNAs for binding in the A-site cleft in the peptidyl-transferase center, which is universally conserved. In addition, these structures support the hypothesis that the species specificity exhibited by the A-site cleft inhibitors is determined by the interactions they make, or fail to make, with a single nucleotide, U2504 (Escherichia coli). In the ribosome, the position of U2504 is controlled by its interactions with neighboring nucleotides, whose identities vary among kingdoms.
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structure of the base of the l7 l12 stalk of the haloarcula marismortui Large Ribosomal Subunit analysis of l11 movements
Journal of Molecular Biology, 2007Co-Authors: Jennifer M Kavran, Thomas A SteitzAbstract:Initiation factors, elongation factors, and release factors all interact with the L7/L12 stalk of the Large Ribosomal Subunit during their respective GTP-dependent cycles on the ribosome. Electron density corresponding to the stalk is not present in previous crystal structures of either 50 S Subunits or 70 S ribosomes. We have now discovered conditions that result in a more ordered factor-binding center in the Haloarcula marismortui (H.ma) Large Ribosomal Subunit crystals and consequently allows the visualization of the full-length L11, the N-terminal domain (NTD) of L10 and helices 43 and 44 of 23 S rRNA. The resulting model is currently the most complete reported structure of a L7/L12 stalk in the context of a ribosome. This region contains a series of intermolecular interfaces that are smaller than those typically seen in other ribonucleoprotein interactions within the 50 S Subunit. Comparisons of the L11 NTD position between the current structure, which is has an NTD splayed out with respect to previous structures, and other structures of ribosomes in different functional states demonstrates a dynamic range of L11 NTD movements. We propose that the L11 NTD moves through three different relative positions during the translational cycle: apo-ribosome, factor-bound pre-GTP hydrolysis and post-GTP hydrolysis. These positions outline a pathway for L11 NTD movements that are dependent on the specific nucleotide state of the bound ligand. These three states are represented by the orientations of the L11 NTD relative to the ribosome and suggest that L11 may play a more specialized role in the factor binding cycle than previously appreciated.
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on the structural basis of peptide bond formation and antibiotic resistance from atomic structures of the Large Ribosomal Subunit
FEBS Letters, 2005Co-Authors: Thomas A SteitzAbstract:The atomic structures of the Large Ribosomal Subunit from Haloarcula marismortui and its complexes with substrates and antibiotics have provided insights into the way the 3000 nucleotide 23S rRNA folds into a compact, specific structure and interacts with 27 Ribosomal proteins as well as the structural basis of the peptidyl transferase reaction and its inhibition by antibiotics. The structure shows that the ribosome is indeed a ribozyme.
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the contribution of metal ions to the structural stability of the Large Ribosomal Subunit
RNA, 2004Co-Authors: D Klein, Peter B. Moore, Thomas A SteitzAbstract:Both monovalent cations and magnesium ions are well known to be essential for the folding and stability of Large RNA molecules that form complex and compact structures. In the atomic structure of the Large Ribosomal Subunit from Haloarcula marismortui, we have identified 116 magnesium ions and 88 monovalent cations bound principally to rRNA. Although the rRNA structures to which these metal ions bind are highly idiosyncratic, a few common principles have emerged from the identities of the specific functional groups that coordinate them. The nonbridging oxygen of a phosphate group is the most common inner shell ligand of Mg++, and Mg++ ions having one or two such inner shell ligands are very common. Nonbridging phosphate oxygens and the heteroatoms of nucleotide bases are common outer shell ligands for Mg++ ions. Monovalent cations usually interact with nucleotide bases and protein groups, although some interactions with nonbridging phosphate oxygens are found. The most common monovalent cation binding site is the major groove side of G-U wobble pairs. Both divalent and monovalent cations stabilize the tertiary structure of 23S rRNA by mediating interactions between its structural domains. Bound metal ions are particularly abundant in the region surrounding the peptidyl transferase center, where stabilizing cationic tails of Ribosomal proteins are notably absent. This may point to the importance of metal ions for the stabilization of specific RNA structures in the evolutionary period prior to the appearance of proteins, and hence many of these metal ion binding sites may be conserved across all phylogenetic kingdoms.
Peter B. Moore - One of the best experts on this subject based on the ideXlab platform.
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Inhibitors of the Large Ribosomal Subunit from Haloarcula marismortui
Israel Journal of Chemistry, 2010Co-Authors: Peter B. MooreAbstract:The crystal structures that have been obtained for 23 different inhibitors bound to the Large Ribosomal Subunit from Haloarcula marismortui are reviewed here. These structures provide important insights into how anti-Ribosomal antibiotics inhibit protein synthesis, how species specificity arises, and the relationship between Ribosomal mutations and antibiotic resistance. These structural studies also provide compelling evidence that the conformation of the peptidyl transferase center of the Large Ribosomal Subunit is intrinsically variable, and that conformational equilibria play a role in determining its functional properties.
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Structures of Triacetyloleandomycin and Mycalamide A Bind to the Large Ribosomal Subunit of Haloarcula marismortui
Antimicrobial Agents and Chemotherapy, 2009Co-Authors: G. Gurel, Thomas A Steitz, Gregor Blaha, Peter B. MooreAbstract:Structures have been obtained for the complexes that triacetyloleandomycin and mycalamide A form with the Large Ribosomal Subunit of Haloarcula marismortui. Triacetyloleandomycin binds in the nascent peptide tunnel and inhibits the activity of ribosomes by blocking the growth of the nascent peptide chain. Mycalamide A binds to the E site and inhibits protein synthesis by occupying the space normally occupied by the CCA end of E-site-bound tRNAs.
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u2504 determines the species specificity of the a site cleft antibiotics the structures of tiamulin homoharringtonine and bruceantin bound to the ribosome
Journal of Molecular Biology, 2009Co-Authors: Guliz Gurel, Peter B. Moore, Thomas A Steitz, Gregor BlahaAbstract:Structures have been obtained for the complexes that tiamulin, homoharringtonine, and bruceantin form with the Large Ribosomal Subunit of Haloarcula marismortui at resolutions ranging from 2.65 to 3.2 A. They show that all these inhibitors block protein synthesis by competing with the amino acid side chains of incoming aminoacyl-tRNAs for binding in the A-site cleft in the peptidyl-transferase center, which is universally conserved. In addition, these structures support the hypothesis that the species specificity exhibited by the A-site cleft inhibitors is determined by the interactions they make, or fail to make, with a single nucleotide, U2504 (Escherichia coli). In the ribosome, the position of U2504 is controlled by its interactions with neighboring nucleotides, whose identities vary among kingdoms.
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the contribution of metal ions to the structural stability of the Large Ribosomal Subunit
RNA, 2004Co-Authors: D Klein, Peter B. Moore, Thomas A SteitzAbstract:Both monovalent cations and magnesium ions are well known to be essential for the folding and stability of Large RNA molecules that form complex and compact structures. In the atomic structure of the Large Ribosomal Subunit from Haloarcula marismortui, we have identified 116 magnesium ions and 88 monovalent cations bound principally to rRNA. Although the rRNA structures to which these metal ions bind are highly idiosyncratic, a few common principles have emerged from the identities of the specific functional groups that coordinate them. The nonbridging oxygen of a phosphate group is the most common inner shell ligand of Mg++, and Mg++ ions having one or two such inner shell ligands are very common. Nonbridging phosphate oxygens and the heteroatoms of nucleotide bases are common outer shell ligands for Mg++ ions. Monovalent cations usually interact with nucleotide bases and protein groups, although some interactions with nonbridging phosphate oxygens are found. The most common monovalent cation binding site is the major groove side of G-U wobble pairs. Both divalent and monovalent cations stabilize the tertiary structure of 23S rRNA by mediating interactions between its structural domains. Bound metal ions are particularly abundant in the region surrounding the peptidyl transferase center, where stabilizing cationic tails of Ribosomal proteins are notably absent. This may point to the importance of metal ions for the stabilization of specific RNA structures in the evolutionary period prior to the appearance of proteins, and hence many of these metal ion binding sites may be conserved across all phylogenetic kingdoms.
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the roles of Ribosomal proteins in the structure assembly and evolution of the Large Ribosomal Subunit
Journal of Molecular Biology, 2004Co-Authors: D Klein, Peter B. Moore, Thomas A SteitzAbstract:The structures of Ribosomal proteins and their interactions with RNA have been examined in the refined crystal structure of the Haloarcula marismortui Large Ribosomal Subunit. The protein structures fall into six groups based on their topology. The 50S Subunit proteins function primarily to stabilize inter-domain interactions that are necessary to maintain the Subunit's structural integrity. An extraordinary variety of protein-RNA interactions is observed. Electrostatic interactions between numerous arginine and lysine residues, particularly those in tail extensions, and the phosphate groups of the RNA backbone mediate many protein-RNA contacts. Base recognition occurs via both the minor groove and widened major groove of RNA helices, as well as through hydrophobic binding pockets that capture bulged nucleotides and through insertion of amino acid residues into hydrophobic crevices in the RNA. Primary binding sites on contiguous RNA are identified for 20 of the 50S Ribosomal proteins, which along with few Large protein-protein interfaces, suggest the order of assembly for some proteins and that the protein extensions fold cooperatively with RNA. The structure supports the hypothesis of co-transcriptional assembly, centered around L24 in domain I. Finally, comparing the structures and locations of the 50S Ribosomal proteins from H.marismortui and D.radiodurans revealed striking examples of molecular mimicry. These comparisons illustrate that identical RNA structures can be stabilized by unrelated proteins.
Roland Beckmann - One of the best experts on this subject based on the ideXlab platform.
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reconstitution of isotopically labeled Ribosomal protein l29 in the 50s Large Ribosomal Subunit for solution state and solid state nmr
Methods of Molecular Biology, 2018Co-Authors: Emeline Barbetmassin, Roland Beckmann, Eli O Van Der Sluis, Joanna Musial, Bernd ReifAbstract:Solid-state nuclear magnetic resonance (NMR) has recently emerged as a method of choice to study structural and dynamic properties of Large biomolecular complexes at atomic resolution. Indeed, recent technological and methodological developments have enabled the study of ever more complex systems in the solid-state. However, to explore multicomponent protein complexes by NMR, specific labeling schemes need to be developed that are dependent on the biological question to be answered. We show here how to reconstitute an isotopically labeled protein within the unlabeled 50S or 70S Ribosomal Subunit. In particular, we focus on the 63-residue Ribosomal protein L29 (~7 kDa), which is located at the exit of the tunnel of the Large 50S Ribosomal Subunit (~1.5 MDa). The aim of this work is the preparation of a suitable sample to investigate allosteric conformational changes in a Ribosomal protein that are induced by the nascent polypeptide chain and that trigger the interaction with different chaperones (e.g., trigger factor or SRP).
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translation regulation via nascent polypeptide mediated ribosome stalling
Current Opinion in Structural Biology, 2016Co-Authors: Daniel N Wilson, Stefan Arenz, Roland BeckmannAbstract:As the nascent polypeptide chain is being synthesized, it passes through a tunnel within the Large Ribosomal Subunit. Interaction between the nascent polypeptide chain and the Ribosomal tunnel can modulate the translation rate and induce translational stalling to regulate gene expression. In this article, we highlight recent structural insights into how the nascent polypeptide chain, either alone or in cooperation with co-factors, can interact with components of the Ribosomal tunnel to regulate translation via inactivating the peptidyltransferase center of the ribosome and inducing ribosome stalling.
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mechanism of eif6 mediated inhibition of Ribosomal Subunit joining
Journal of Biological Chemistry, 2010Co-Authors: Marco Gartmann, Michael Blau, Jeanpaul Armache, Thorsten Mielke, Maya Topf, Roland BeckmannAbstract:During the process of Ribosomal assembly, the essential eukaryotic translation initiation factor 6 (eIF6) is known to act as a Ribosomal anti-association factor. However, a molecular understanding of the anti-association activity of eIF6 is still missing. Here we present the cryo-electron microscopy reconstruction of a complex of the Large Ribosomal Subunit with eukaryotic eIF6 from Saccharomyces cerevisiae. The structure reveals that the eIF6 binding site involves mainly rpL23 (L14p in Escherichia coli). Based on our structural data, we propose that the mechanism of the anti-association activity of eIF6 is based on steric hindrance of interSubunit bridge formation around the dynamic bridge B6.
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architecture of the protein conducting channel associated with the translating 80s ribosome
Cell, 2001Co-Authors: Piotr Andrzej Penczek, Joachim Frank, Roland Beckmann, Christian M T Spahn, Narayanan Eswar, Jurgen Helmers, Andrej SaliAbstract:In vitro assembled yeast ribosome-nascent chain complexes (RNCs) containing a signal sequence in the nascent chain were immunopurified and reconstituted with the purified protein-conducting channel (PCC) of yeast endoplasmic reticulum, the Sec61 complex. A cryo-EM reconstruction of the RNC-Sec61 complex at 15.4 A resolution shows a tRNA in the P site. Distinct rRNA elements and proteins of the Large Ribosomal Subunit form four connections with the PCC across a gap of about 10-20 A. Binding of the PCC influences the position of the highly dynamic rRNA expansion segment 27. The RNC-bound Sec61 complex has a compact appearance and was estimated to be a trimer. We propose a binary model of cotranslational translocation entailing only two basic functional states of the translating ribosome-channel complex.
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alignment of conduits for the nascent polypeptide chain in the ribosome sec61 complex
Science, 1997Co-Authors: Roland Beckmann, Piotr Andrzej Penczek, Robert A Grassucci, Doryen Bubeck, Adriana Verschoor, Gunter Blobel, Joachim FrankAbstract:An oligomer of the Sec61 trimeric complex is thought to form the protein-conducting channel for protein transport across the endoplasmic reticulum. A purified yeast Sec61 complex bound to monomeric yeast ribosomes as an oligomer in a saturable fashion. Cryo-electron microscopy of the ribosome-Sec61 complex and a three-dimensional reconstruction showed that the Sec61 oligomer is attached to the Large Ribosomal Subunit by a single connection. Moreover, a funnel-shaped pore in the Sec61 oligomer aligned with the exit of a tunnel traversing the Large Ribosomal Subunit, strongly suggesting that both structures function together in the translocation of proteins across the endoplasmic reticulum membrane.
Steven Clarke - One of the best experts on this subject based on the ideXlab platform.
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histidine methylation of yeast Ribosomal protein rpl3p is required for proper 60s Subunit assembly
Molecular and Cellular Biology, 2014Co-Authors: Qais Alhadid, Kevin Roy, Guillaume Chanfreau, William Munroe, Maria C Dzialo, Steven ClarkeAbstract:Histidine protein methylation is an unusual posttranslational modification. In the yeast Saccharomyces cerevisiae, the Large Ribosomal Subunit protein Rpl3p is methylated at histidine 243, a residue that contacts the 25S rRNA near the P site. Rpl3p methylation is dependent upon the presence of Hpm1p, a candidate seven-beta-strand methyltransferase. In this study, we elucidated the biological activities of Hpm1p in vitro and in vivo. Amino acid analyses reveal that Hpm1p is responsible for all of the detectable protein histidine methylation in yeast. The modification is found on a polypeptide corresponding to the size of Rpl3p in ribosomes and in a nucleus-containing organelle fraction but was not detected in proteins of the ribosome-free cytosol fraction. In vitro assays demonstrate that Hpm1p has methyltransferase activity on ribosome-associated but not free Rpl3p, suggesting that its activity depends on interactions with Ribosomal components. hpm1 null cells are defective in early rRNA processing, resulting in a deficiency of 60S Subunits and translation initiation defects that are exacerbated in minimal medium. Cells lacking Hpm1p are resistant to cycloheximide and verrucarin A and have decreased translational fidelity. We propose that Hpm1p plays a role in the orchestration of the early assembly of the Large Ribosomal Subunit and in faithful protein production.
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histidine methylation of yeast Ribosomal protein rpl3p is required for proper 60s Subunit assembly lb185
The FASEB Journal, 2014Co-Authors: Qais Alhadid, Kevin Roy, Guillaume Chanfreau, Steven ClarkeAbstract:Histidine protein methylation is an unusual posttranslational modification. In the yeast Saccharomyces cerevisiae, the Large Ribosomal Subunit protein Rpl3p is methylated at histidine-243, a residu...
Ada Yonath - One of the best experts on this subject based on the ideXlab platform.
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a novel pleuromutilin antibacterial compound its binding mode and selectivity mechanism
Scientific Reports, 2016Co-Authors: Z Eyal, Susanne Paukner, Rosemarie Riedl, Anat Bashan, Donna Matzov, Miri Krupkin, Haim Rozenberg, Ella Zimmerman, Ada YonathAbstract:The increasing appearance of pathogenic bacteria with antibiotic resistance is a global threat. Consequently, clinically available potent antibiotics that are active against multidrug resistant pathogens are becoming exceedingly scarce. Ribosomes are a main target for antibiotics, and hence are an objective for novel drug development. Lefamulin, a semi-synthetic pleuromutilin compound highly active against multi-resistant pathogens, is a promising antibiotic currently in phase III trials for the treatment of community-acquired bacterial pneumonia in adults. The crystal structure of the Staphylococcus aureus Large Ribosomal Subunit in complex with lefamulin reveals its protein synthesis inhibition mechanism and the rationale for its potency. In addition, analysis of the bacterial and eukaryotes ribosome structures around the pleuromutilin binding pocket has elucidated the key for the drug’s selectivity.
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2 8 a cryo em structure of the Large Ribosomal Subunit from the eukaryotic parasite leishmania
Cell Reports, 2016Co-Authors: Moran Shalevbenami, Anat Bashan, Donna Matzov, Haim Rozenberg, Ella Zimmerman, Yan Zhang, Y Halfon, Arie Zackay, Charles L Jaffe, Ada YonathAbstract:Summary Leishmania is a single-cell eukaryotic parasite of the Trypanosomatidae family, whose members cause an array of tropical diseases. The often fatal outcome of infections, lack of effective vaccines, limited selection of therapeutic drugs, and emerging resistant strains, underline the need to develop strategies to combat these pathogens. The Trypanosomatid ribosome has recently been highlighted as a promising therapeutic target due to structural features that are distinct from other eukaryotes. Here, we present the 2.8-A resolution structure of the Leishmania donovani Large Ribosomal Subunit (LSU) derived from a cryo-EM map, further enabling the structural observation of eukaryotic rRNA modifications that play a significant role in ribosome assembly and function. The structure illustrates the unique fragmented nature of leishmanial LSU rRNA and highlights the irregular distribution of rRNA modifications in Leishmania, a characteristic with implications for anti-parasitic drug development.
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structural insight into the antibiotic action of telithromycin against resistant mutants
Journal of Bacteriology, 2003Co-Authors: Rita Berisio, Frank Schluenzen, Joerg Harms, Paola Fucini, Raz Zarivach, Harly A S Hansen, Ada YonathAbstract:The crystal structure of the ketolide telithromycin bound to the Deinococcus radiodurans Large Ribosomal Subunit shows that telithromycin blocks the Ribosomal exit tunnel and interacts with domains II and V of the 23S RNA. Comparisons to other clinically relevant macrolides provided structural insights into its enhanced activity against macrolide-resistant strains.
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high resolution structure of the Large Ribosomal Subunit from a mesophilic eubacterium
Cell, 2001Co-Authors: Joerg Harms, Heike Bartels, Anat Bashan, Frank Schluenzen, Ilana Agmon, Sharon Gat, Francois Franceschi, Ada YonathAbstract:We describe the high resolution structure of the Large Ribosomal Subunit from Deinococcus radiodurans (D50S), a gram-positive mesophile suitable for binding of antibiotics and functionally relevant ligands. The over-all structure of D50S is similar to that from the archae bacterium Haloarcula marismortui (H50S); however, a detailed comparison revealed significant differences, for example, in the orientation of nucleotides in peptidyl transferase center and in the structures of many Ribosomal proteins. Analysis of Ribosomal features involved in dynamic aspects of protein biosynthesis that are partially or fully disordered in H50S revealed the conformations of interSubunit bridges in unbound Subunits, suggesting how they may change upon Subunit association and how movements of the L1-stalk may facilitate the exit of tRNA.
