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Roger A Garrett - One of the best experts on this subject based on the ideXlab platform.

  • puromycin rrna interaction sites at the Peptidyl Transferase center
    RNA, 2000
    Co-Authors: Cristina Rodriguezfonseca, S V Kirillov, Hien Phan, Katherine S Long, Bo T Porse, Ricardo Amils, Roger A Garrett
    Abstract:

    The binding site of puromycin was probed chemically in the Peptidyl-Transferase center of ribosomes from Escherichia coli and of puromycin-hypersensitive ribosomes from the archaeon Haloferax gibbonsii. Several nucleotides of the 23S rRNAs showed altered chemical reactivities in the presence of puromycin. They include A2439, G2505, and G2553 for E. coli, and G2058, A2503, G2505, and G2553 for Hf. gibbonsii (using the E. coli numbering system). Reproducible enhanced reactivities were also observed at A508 and A1579 within domains I and III, respectively, of E. coli 23S rRNA. In further experiments, puromycin was shown to produce a major reduction in the UV-induced crosslinking of deacylated-(2N3A76)tRNA to U2506 within the P9 site of E. coli ribosomes. Moreover, it strongly stimulated the putative UV-induced crosslink between a streptogramin B drug and m 2 A2503/C2504 at an adjacent site in E. coli 23S rRNA. These data strongly support the concept that puromycin, along with other Peptidyl-Transferase antibiotics, in particular the streptogramin B drugs, bind to an RNA structural motif that contains several conserved and accessible base moieties of the Peptidyl Transferase loop region. This streptogramin motif is also likely to provide binding sites for the 39 termini of the acceptor and donor tRNAs. In contrast, the effects at A508 and A1579, which are located at the exit site of the peptide channel, are likely to be caused by a structural effect transmitted along the peptide channel.

  • uv induced modifications in the Peptidyl Transferase loop of 23s rrna dependent on binding of the streptogramin b antibiotic pristinamycin ia
    RNA, 1999
    Co-Authors: Bo T Porse, S V Kirillov, Mariana J Awayez, Roger A Garrett
    Abstract:

    The naturally occurring streptogramin B antibiotic, pristinamycin IA, which inhibits peptide elongation, can produce two modifications in 23S rRNA when bound to the Escherichia coli 70S ribosome and irradiated at 365 nm. Both drug-induced effects map to highly conserved nucleotides within the functionally important Peptidyl Transferase loop of 23S rRNA at positions m2A2503/psi2504 and G2061/A2062. The modification yields are influenced strongly, and differentially, by P-site-bound tRNA and strongly by some of the Peptidyl Transferase antibiotics tested, with chloramphenicol producing a shift in the latter modification to A2062/C2063. Pristinamycin IA can also produce a modification on binding to deproteinized, mature 23S rRNA, at position U2500/C2501. The same modification occurs on an approximately 37-nt fragment, encompassing positions approximately 2496-2532 of the Peptidyl Transferase loop that was excised from the mature rRNA using RNAse H. In contrast, no antibiotic-induced effects were observed on in vitro T7 transcripts of full-length 23S rRNA, domain V, or on a fragment extending from positions approximately 2496-2566, which indicates that one or more posttranscriptional modifications within the sequence Cm-C-U-C-G-m2A-psi-G2505 are important for pristinamycin IA binding and/or the antibiotic-dependent modification of 23S rRNA.

  • movement of the 3 end of trna through the Peptidyl Transferase centre and its inhibition by antibiotics
    FEBS Letters, 1997
    Co-Authors: S V Kirillov, Bo T Porse, Birte Vester, Paul Woolley, Roger A Garrett
    Abstract:

    Determining how antibiotics inhibit ribosomal activity requires a detailed understanding of the interactions and relative movement of tRNA, mRNA and the ribosome. Recent models for the formation of hybrid tRNA binding sites during the elongation cycle have provided a basis for re-evaluating earlier experimental data and, especially, those relevant to substrate movements through the Peptidyl Transferase centre. With the exception of deacylated tRNA, which binds at the E-site, ribosomal interactions of the 3′-ends of the tRNA substrates generate only a small part of the total free energy of tRNA-ribosome binding. Nevertheless, these relatively weak interactions determine the unidirectional movement of tRNAs through the ribosome and, moreover, they appear to be particularly susceptible to perturbation by antibiotics. Here we summarise current ideas relating particularly to the movement of the 3′-ends of tRNA through the ribosome and consider possible inhibitory mechanisms of the Peptidyl Transferase antibiotics.

  • Movement of the 3′-end of tRNA through the Peptidyl Transferase centre and its inhibition by antibiotics
    FEBS Letters, 1997
    Co-Authors: S V Kirillov, Bo T Porse, Birte Vester, Paul Woolley, Roger A Garrett
    Abstract:

    Determining how antibiotics inhibit ribosomal activity requires a detailed understanding of the interactions and relative movement of tRNA, mRNA and the ribosome. Recent models for the formation of hybrid tRNA binding sites during the elongation cycle have provided a basis for re-evaluating earlier experimental data and, especially, those relevant to substrate movements through the Peptidyl Transferase centre. With the exception of deacylated tRNA, which binds at the E-site, ribosomal interactions of the 3′-ends of the tRNA substrates generate only a small part of the total free energy of tRNA-ribosome binding. Nevertheless, these relatively weak interactions determine the unidirectional movement of tRNAs through the ribosome and, moreover, they appear to be particularly susceptible to perturbation by antibiotics. Here we summarise current ideas relating particularly to the movement of the 3′-ends of tRNA through the ribosome and consider possible inhibitory mechanisms of the Peptidyl Transferase antibiotics.

  • a sparsomycin resistant mutant ofhalobacterium salinariumlacks a modification at nucleotide u2603 in the Peptidyl Transferase centre of 23 s rrna
    Journal of Molecular Biology, 1996
    Co-Authors: Ester Lazaro, Cristina Rodriguezfonseca, Bo T Porse, Roger A Garrett, Dionisio Urena, Juan P. G. Ballesta
    Abstract:

    Sparsomycin, a broad-spectrum antibiotic, acts at the Peptidyl Transferase centre of the ribosome, stabilizing Peptidyl-tRNA binding at the P-site and weakening ternary complex binding. A sparsomycin-resistant mutant was isolated for the archaeonHalobacterium salinariumand shown to lack a post-transcriptional modification of U2603 (Escherichia colinumbering U2584), which is a universally conserved uridine base located within the Peptidyl Transferase loop of 23 S rRNA. This mutant also exhibited altered sensitivities to the Peptidyl Transferase antibiotics anisomycin, chloram phenicol and puromycin. Several lines of evidence indicate that the unmodified uridine base lies within the P-substrate site of the Peptidyl Transferase centre.

Alexander S Mankin - One of the best experts on this subject based on the ideXlab platform.

  • context specific inhibition of translation by ribosomal antibiotics targeting the Peptidyl Transferase center
    Proceedings of the National Academy of Sciences of the United States of America, 2016
    Co-Authors: James Marks, Krishna Kannan, Emily J Roncase, Dorota Klepacki, Amira Kefi, Cedric Orelle, Nora Vazquezlaslop, Alexander S Mankin
    Abstract:

    The first broad-spectrum antibiotic chloramphenicol and one of the newest clinically important antibacterials, linezolid, inhibit protein synthesis by targeting the Peptidyl Transferase center of the bacterial ribosome. Because antibiotic binding should prevent the placement of aminoacyl-tRNA in the catalytic site, it is commonly assumed that these drugs are universal inhibitors of Peptidyl transfer and should readily block the formation of every peptide bond. However, our in vitro experiments showed that chloramphenicol and linezolid stall ribosomes at specific mRNA locations. Treatment of bacterial cells with high concentrations of these antibiotics leads to preferential arrest of translation at defined sites, resulting in redistribution of the ribosomes on mRNA. Antibiotic-mediated inhibition of protein synthesis is most efficient when the nascent peptide in the ribosome carries an alanine residue and, to a lesser extent, serine or threonine in its penultimate position. In contrast, the inhibitory action of the drugs is counteracted by glycine when it is either at the nascent-chain C terminus or at the incoming aminoacyl-tRNA. The context-specific action of chloramphenicol illuminates the operation of the mechanism of inducible resistance that relies on programmed drug-induced translation arrest. In addition, our findings expose the functional interplay between the nascent chain and the Peptidyl Transferase center.

  • an indigenous posttranscriptional modification in the ribosomal Peptidyl Transferase center confers resistance to an array of protein synthesis inhibitors
    Journal of Molecular Biology, 2008
    Co-Authors: Alexander S Mankin
    Abstract:

    A number of nucleotide residues in ribosomal RNA (rRNA) undergo specific posttranscriptional modifications. The roles of most modifications are unclear, but their clustering in functionally important regions of rRNA suggests that they might either directly affect the activity of the ribosome or modulate its interactions with ligands. Of the 25 modified nucleotides in Escherichia coli 23S rRNA, 14 are located in the Peptidyl Transferase center, the main antibiotic target in the large ribosomal subunit. Since nucleotide modifications have been closely associated with both antibiotic sensitivity and antibiotic resistance, loss of some of these posttranscriptional modifications may affect the susceptibility of bacteria to antibiotics. We investigated the antibiotic sensitivity of E. coli cells in which the genes of 8 rRNA-modifying enzymes targeting the Peptidyl Transferase center were individually inactivated. The lack of pseudouridine at position 2504 of 23S rRNA was found to significantly increase the susceptibility of bacteria to Peptidyl Transferase inhibitors. Therefore, this indigenous posttranscriptional modification may have evolved as an intrinsic resistance mechanism protecting bacteria against natural antibiotics.

  • spark a new Peptidyl Transferase activity assay
    Methods in molecular medicine, 2008
    Co-Authors: Alexander S Mankin, Norbert Polacek
    Abstract:

    : The formation of peptide bonds is the central chemical reaction during protein synthesis and is catalyzed by the Peptidyl Transferase center residing in the large ribosomal subunit. This active site is composed of universally conserved rRNA nucleosides. The Peptidyl Transferase center is by far the most frequently used target site of natural antibiotics in the cell. Here we describe a novel, simple, and convenient method to assess peptide bond formation which we named SPARK. The basic principle of SPARK is the use of two reaction substrates that closely resemble the natural tRNA substrates (one is biotinylated and the other carries a tritium label) that become covalently connected during transpeptidation. Formation of this peptide bond then allows capture and direct quantification of the radiolabled product, now joined to the biotin group, using the scintillation proximity assay technology. Binding of the tritiated radioligand to streptavidin-coated beads causes the excitation of the bead-embedded scintillant, thus resulting in the detection of radioactivity. Since no product purification step is required, SPARK is amenable to simple automation, which makes it useful in high-throughput screens of natural or synthetic compound libraries in the search for novel antibiotics.

  • the ribosomal Peptidyl Transferase center structure function evolution inhibition
    Critical Reviews in Biochemistry and Molecular Biology, 2005
    Co-Authors: Norbert Polacek, Alexander S Mankin
    Abstract:

    ABSTRACTThe ribosomal Peptidyl Transferase center (PTC) resides in the large ribosomal subunit and catalyzes the two principal chemical reactions of protein synthesis: peptide bond formation and peptide release. The catalytic mechanisms employed and their inhibition by antibiotics have been in the focus of molecular and structural biologists for decades. With the elucidation of atomic structures of the large ribosomal subunit at the dawn of the new millennium, these questions gained a new level of molecular significance. The crystallographic structures compellingly confirmed that Peptidyl Transferase is an RNA enzyme. This places the ribosome on the list of naturally occurring riboyzmes that outlived the transition from the pre-biotic RNA World to contemporary biology. Biochemical, genetic and structural evidence highlight the role of the ribosome as an entropic catalyst that accelerates peptide bond formation primarily by substrate positioning. At the same time, peptide release should more strongly depen...

  • Chemical engineering of the Peptidyl Transferase center reveals an important role of the 2′-hydroxyl group of A2451
    Nucleic Acids Research, 2005
    Co-Authors: Matthias D Erlacher, Alexander S Mankin, Kathrin Lang, Nisha Shankaran, Brigitte Wotzel, Alexander Huttenhofer, Ronald Micura, Norbert Polacek
    Abstract:

    The main enzymatic reaction of the large ribosomal subunit is peptide bond formation. Ribosome crystallography showed that A2451 of 23S rRNA makes the closest approach to the attacking amino group of aminoacyl-tRNA. Mutations of A2451 had relatively small effects on transpeptidation and failed to unequivocally identify the crucial functional group(s). Here, we employed an in vitro reconstitution system for chemical engineering the Peptidyl Transferase center by introducing non-natural nucleosides at position A2451. This allowed us to investigate the Peptidyl transfer reaction performed by a ribosome that contained a modified nucleoside at the active site. The main finding is that ribosomes carrying a 2 0 -deoxyribose at A2451 showed a compromised Peptidyl Transferase activity. In variance, adenine base modifications and even the removal of the entire nucleobase at A2451 had only little impact on peptide bond formation, as long as the 2 0 -hydroxyl was present. This implicates a functional or structural role of the 2 0 -hydroxyl group at A2451 for transpeptidation.

Norbert Polacek - One of the best experts on this subject based on the ideXlab platform.

  • the role of the universally conserved a2450 c2063 base pair in the ribosomal Peptidyl Transferase center
    Nucleic Acids Research, 2010
    Co-Authors: Anna Chirkova, Matthias D Erlacher, Nina Clementi, Marek Zywicki, Michaela Aigner, Norbert Polacek
    Abstract:

    : Despite the fact that all 23S rRNA nucleotides that build the ribosomal Peptidyl Transferase ribozyme are universally conserved, standard and atomic mutagenesis studies revealed the nucleobase identities being non-critical for catalysis. This indicates that these active site residues are highly conserved for functions distinct from catalysis. To gain insight into potential contributions, we have manipulated the nucleobases via an atomic mutagenesis approach and have utilized these chemically engineered ribosomes for in vitro translation reactions. We show that most of the active site nucleobases could be removed without significant effects on polypeptide production. Our data however highlight the functional importance of the universally conserved non-Watson-Crick base pair at position A2450-C2063. Modifications that disrupt this base pair markedly impair translation activities, while having little effects on peptide bond formation, tRNA drop-off and ribosome-dependent EF-G GTPase activity. Thus it seems that disruption of the A2450-C2063 pair inhibits a reaction following transpeptidation and EF-G action during the elongation cycle. Cumulatively our data are compatible with the hypothesis that the integrity of this A-C wobble base pair is essential for effective tRNA translocation through the Peptidyl Transferase center during protein synthesis.

  • The role of the universally conserved A2450–C2063 base pair in the ribosomal Peptidyl Transferase center
    Nucleic Acids Research, 2010
    Co-Authors: Anna Chirkova, Matthias D Erlacher, Nina Clementi, Marek Zywicki, Michaela Aigner, Norbert Polacek
    Abstract:

    : Despite the fact that all 23S rRNA nucleotides that build the ribosomal Peptidyl Transferase ribozyme are universally conserved, standard and atomic mutagenesis studies revealed the nucleobase identities being non-critical for catalysis. This indicates that these active site residues are highly conserved for functions distinct from catalysis. To gain insight into potential contributions, we have manipulated the nucleobases via an atomic mutagenesis approach and have utilized these chemically engineered ribosomes for in vitro translation reactions. We show that most of the active site nucleobases could be removed without significant effects on polypeptide production. Our data however highlight the functional importance of the universally conserved non-Watson-Crick base pair at position A2450-C2063. Modifications that disrupt this base pair markedly impair translation activities, while having little effects on peptide bond formation, tRNA drop-off and ribosome-dependent EF-G GTPase activity. Thus it seems that disruption of the A2450-C2063 pair inhibits a reaction following transpeptidation and EF-G action during the elongation cycle. Cumulatively our data are compatible with the hypothesis that the integrity of this A-C wobble base pair is essential for effective tRNA translocation through the Peptidyl Transferase center during protein synthesis.

  • spark a new Peptidyl Transferase activity assay
    Methods in molecular medicine, 2008
    Co-Authors: Alexander S Mankin, Norbert Polacek
    Abstract:

    : The formation of peptide bonds is the central chemical reaction during protein synthesis and is catalyzed by the Peptidyl Transferase center residing in the large ribosomal subunit. This active site is composed of universally conserved rRNA nucleosides. The Peptidyl Transferase center is by far the most frequently used target site of natural antibiotics in the cell. Here we describe a novel, simple, and convenient method to assess peptide bond formation which we named SPARK. The basic principle of SPARK is the use of two reaction substrates that closely resemble the natural tRNA substrates (one is biotinylated and the other carries a tritium label) that become covalently connected during transpeptidation. Formation of this peptide bond then allows capture and direct quantification of the radiolabled product, now joined to the biotin group, using the scintillation proximity assay technology. Binding of the tritiated radioligand to streptavidin-coated beads causes the excitation of the bead-embedded scintillant, thus resulting in the detection of radioactivity. Since no product purification step is required, SPARK is amenable to simple automation, which makes it useful in high-throughput screens of natural or synthetic compound libraries in the search for novel antibiotics.

  • the ribosomal Peptidyl Transferase center structure function evolution inhibition
    Critical Reviews in Biochemistry and Molecular Biology, 2005
    Co-Authors: Norbert Polacek, Alexander S Mankin
    Abstract:

    ABSTRACTThe ribosomal Peptidyl Transferase center (PTC) resides in the large ribosomal subunit and catalyzes the two principal chemical reactions of protein synthesis: peptide bond formation and peptide release. The catalytic mechanisms employed and their inhibition by antibiotics have been in the focus of molecular and structural biologists for decades. With the elucidation of atomic structures of the large ribosomal subunit at the dawn of the new millennium, these questions gained a new level of molecular significance. The crystallographic structures compellingly confirmed that Peptidyl Transferase is an RNA enzyme. This places the ribosome on the list of naturally occurring riboyzmes that outlived the transition from the pre-biotic RNA World to contemporary biology. Biochemical, genetic and structural evidence highlight the role of the ribosome as an entropic catalyst that accelerates peptide bond formation primarily by substrate positioning. At the same time, peptide release should more strongly depen...

  • Chemical engineering of the Peptidyl Transferase center reveals an important role of the 2′-hydroxyl group of A2451
    Nucleic Acids Research, 2005
    Co-Authors: Matthias D Erlacher, Alexander S Mankin, Kathrin Lang, Nisha Shankaran, Brigitte Wotzel, Alexander Huttenhofer, Ronald Micura, Norbert Polacek
    Abstract:

    The main enzymatic reaction of the large ribosomal subunit is peptide bond formation. Ribosome crystallography showed that A2451 of 23S rRNA makes the closest approach to the attacking amino group of aminoacyl-tRNA. Mutations of A2451 had relatively small effects on transpeptidation and failed to unequivocally identify the crucial functional group(s). Here, we employed an in vitro reconstitution system for chemical engineering the Peptidyl Transferase center by introducing non-natural nucleosides at position A2451. This allowed us to investigate the Peptidyl transfer reaction performed by a ribosome that contained a modified nucleoside at the active site. The main finding is that ribosomes carrying a 2 0 -deoxyribose at A2451 showed a compromised Peptidyl Transferase activity. In variance, adenine base modifications and even the removal of the entire nucleobase at A2451 had only little impact on peptide bond formation, as long as the 2 0 -hydroxyl was present. This implicates a functional or structural role of the 2 0 -hydroxyl group at A2451 for transpeptidation.

Birte Vester - One of the best experts on this subject based on the ideXlab platform.

  • the pleuromutilin drugs tiamulin and valnemulin bind to the rna at the Peptidyl Transferase centre on the ribosome
    Molecular Microbiology, 2008
    Co-Authors: Susan M Poulsen, M Karlsson, Lena B Johansson, Birte Vester
    Abstract:

    : The pleuromutilin antibiotic derivatives, tiamulin and valnemulin, inhibit protein synthesis by binding to the 50S ribosomal subunit of bacteria. The action and binding site of tiamulin and valnemulin was further characterized on Escherichia coli ribosomes. It was revealed that these drugs are strong inhibitors of Peptidyl Transferase and interact with domain V of 23S RNA, giving clear chemical footprints at nucleotides A2058-9, U2506 and U2584-5. Most of these nucleotides are highly conserved phylogenetically and functionally important, and all of them are at or near the Peptidyl Transferase centre and have been associated with binding of several antibiotics. Competitive footprinting shows that tiamulin and valnemulin can bind concurrently with the macrolide erythromycin but compete with the macrolide carbomycin, which is a Peptidyl Transferase inhibitor. We infer from these and previous results that tiamulin and valnemulin interact with the rRNA in the Peptidyl Transferase slot on the ribosomes in which they prevent the correct positioning of the CCA-ends of tRNAs for peptide transfer.

  • interaction of pleuromutilin derivatives with the ribosomal Peptidyl Transferase center
    Antimicrobial Agents and Chemotherapy, 2006
    Co-Authors: Katherine S Long, Lykke Haastrup Hansen, Lene Jakobsen, Birte Vester
    Abstract:

    Tiamulin is a pleuromutilin antibiotic that is used in veterinary medicine. The recently published crystal structure of a tiamulin-50S ribosomal subunit complex provides detailed information about how this drug targets the Peptidyl Transferase center of the ribosome. To promote rational design of pleuromutilin-based drugs, the binding of the antibiotic pleuromutilin and three semisynthetic derivatives with different side chain extensions has been investigated using chemical footprinting. The nucleotides A2058, A2059, G2505, and U2506 are affected in all of the footprints, suggesting that the drugs are similarly anchored in the binding pocket by the common tricyclic mutilin core. However, varying effects are observed at U2584 and U2585, indicating that the side chain extensions adopt distinct conformations within the cavity and thereby affect the rRNA conformation differently. An Escherichia coli L3 mutant strain is resistant to tiamulin and pleuromutilin, but not valnemulin, implying that valnemulin is better able to withstand an altered rRNA binding surface around the mutilin core. This is likely due to additional interactions made between the valnemulin side chain extension and the rRNA binding site. The data suggest that pleuromutilin drugs with enhanced antimicrobial activity may be obtained by maximizing the number of interactions between the side chain moiety and the Peptidyl Transferase cavity.

  • resistance to the Peptidyl Transferase inhibitor tiamulin caused by mutation of ribosomal protein l3
    Antimicrobial Agents and Chemotherapy, 2003
    Co-Authors: Jacob Bosling, Birte Vester, Susan M Poulsen, Katherine S Long
    Abstract:

    The antibiotic tiamulin targets the 50S subunit of the bacterial ribosome and interacts at the Peptidyl Transferase center. Tiamulin-resistant Escherichia coli mutants were isolated in order to elucidate mechanisms of resistance to the drug. No mutations in the rRNA were selected as resistance determinants using a strain expressing only a plasmid-encoded rRNA operon. Selection in a strain with all seven chromosomal rRNA operons yielded a mutant with an A445G mutation in the gene coding for ribosomal protein L3, resulting in an Asn149Asp alteration. Complementation experiments and sequencing of transductants demonstrate that the mutation is responsible for the resistance phenotype. Chemical footprinting experiments show a reduced binding of tiamulin to mutant ribosomes. It is inferred that the L3 mutation, which points into the Peptidyl Transferase cleft, causes tiamulin resistance by alteration of the drug-binding site. This is the first report of a mechanism of resistance to tiamulin unveiled in molecular detail.

  • inhibition of the ribosomal Peptidyl Transferase reaction by the mycarose moiety of the antibiotics carbomycin spiramycin and tylosin
    Journal of Molecular Biology, 2000
    Co-Authors: Susan M Poulsen, Christine Kofoed, Birte Vester
    Abstract:

    Many antibiotics, including the macrolides, inhibit protein synthesis by binding to ribosomes. Only some of the macrolides affect the Peptidyl Transferase reaction. The 16-member ring macrolide antibiotics carbomycin, spiramycin, and tylosin inhibit Peptidyl Transferase. All these have a disaccharide at position 5 in the lactone ring with a mycarose moiety. We have investigated the functional role of this mycarose moiety. The 14-member ring macrolide erythromycin and the 16-member ring macrolides desmycosin and chalcomycin do not inhibit the Peptidyl Transferase reaction. These drugs have a monosaccharide at position 5 in the lactone ring. The presence of mycarose was correlated with inhibition of Peptidyl Transferase, footprints on 23 S rRNA and whether the macrolide can compete with binding of hygromycin A to the ribosome. The binding sites of the macrolides to Escherichia coli ribosomes were investigated by chemical probing of domains II and V of 23 S rRNA. The common binding site is around position A2058, while effects on U2506 depend on the presence of the mycarose sugar. Also, protection at position A752 indicates that a mycinose moiety at position 14 in 16-member ring macrolides interact with hairpin 35 in domain II. Competitive footprinting of ribosomal binding of hygromycin A and macrolides showed that tylosin and spiramycin reduce the hygromycin A protections of nucleotides in 23 S rRNA and that carbomycin abolishes its binding. In contrast, the macrolides that do not inhibit the Peptidyl Transferase reaction bind to the ribosomes concurrently with hygromycin A. Data are presented to argue that a disaccharide at position 5 in the lactone ring of macrolides is essential for inhibition of peptide bond formation and that the mycarose moiety is placed near the conserved U2506 in the central loop region of domain V 23 S rRNA.

  • movement of the 3 end of trna through the Peptidyl Transferase centre and its inhibition by antibiotics
    FEBS Letters, 1997
    Co-Authors: S V Kirillov, Bo T Porse, Birte Vester, Paul Woolley, Roger A Garrett
    Abstract:

    Determining how antibiotics inhibit ribosomal activity requires a detailed understanding of the interactions and relative movement of tRNA, mRNA and the ribosome. Recent models for the formation of hybrid tRNA binding sites during the elongation cycle have provided a basis for re-evaluating earlier experimental data and, especially, those relevant to substrate movements through the Peptidyl Transferase centre. With the exception of deacylated tRNA, which binds at the E-site, ribosomal interactions of the 3′-ends of the tRNA substrates generate only a small part of the total free energy of tRNA-ribosome binding. Nevertheless, these relatively weak interactions determine the unidirectional movement of tRNAs through the ribosome and, moreover, they appear to be particularly susceptible to perturbation by antibiotics. Here we summarise current ideas relating particularly to the movement of the 3′-ends of tRNA through the ribosome and consider possible inhibitory mechanisms of the Peptidyl Transferase antibiotics.

Scott A Strobel - One of the best experts on this subject based on the ideXlab platform.

  • transition state chirality and role of the vicinal hydroxyl in the ribosomal Peptidyl Transferase reaction
    Biochemistry, 2008
    Co-Authors: Kevin S Huang, Nicolas Carrasco, Emmanuel Pfund, Scott A Strobel
    Abstract:

    The ribosomal Peptidyl Transferase is a biologically essential catalyst responsible for protein synthesis. The reaction is expected to proceed through a transition state approaching tetrahedral geometry with a specific chirality. To establish that stereospecificity, we synthesized two diastereomers of a transition state inhibitor with mimics for each of the four ligands around the reactive chiral center. Preferential binding of the inhibitor that mimics a transition state with S chirality establishes the spatial position of the nascent peptide, the oxyanion and places the amine near the critical A76 2′-OH on the P-site tRNA. Another inhibitor series with 2′-NH2 and 2′-SH substitutions at the critical 2′-OH group was used to test the neutrality of the 2′-OH as predicted if the hydroxyl functions as a proton shuttle in the transition state. Lack of significant pH dependent binding by these inhibitors argues that the 2′-OH remains neutral in the transition state. Both of these observations are consistent with a proton shuttle mechanism for the Peptidyl Transferase reaction.

  • structural insights into the roles of water and the 2 hydroxyl of the p site trna in the Peptidyl Transferase reaction
    Molecular Cell, 2005
    Co-Authors: T M Schmeing, Thomas A Steitz, Scott A Strobel, Kevin S Huang, David Kitchen
    Abstract:

    Summary Peptide bond formation is catalyzed at the Peptidyl Transferase center (PTC) of the large ribosomal subunit. Crystal structures of the large ribosomal subunit of Haloarcula marismortui (Hma) complexed with several analogs that represent either the substrates or the transition state intermediate of the Peptidyl Transferase reaction show that this reaction proceeds through a tetrahedral intermediate with S chirality. The oxyanion of the tetrahedral intermediate interacts with a water molecule that is positioned by nucleotides A2637 ( E. coli numbering, 2602) and methyl U2619(2584). There are no Mg 2+ ions or monovalent metal ions observed in the PTC that could directly promote catalysis. The A76 2′ hydroxyl of the Peptidyl-tRNA is hydrogen bonded to the α-amino group and could facilitate peptide bond formation by substrate positioning and by acting as a proton shuttle between the α-amino group and the A76 3′ hydroxyl of the Peptidyl-tRNA.

  • uncovering the enzymatic pka of the ribosomal Peptidyl Transferase reaction utilizing a fluorinated puromycin derivative
    Biochemistry, 2005
    Co-Authors: Kensuke Okuda, Amy C Seila, Scott A Strobel
    Abstract:

    The ribosome-catalyzed Peptidyl Transferase reaction displays a complex pH profile resulting from two functional groups whose deprotonation is important for the reaction, one within the A-site subs...

  • ph dependent conformational flexibility within the ribosomal Peptidyl Transferase center
    RNA, 2001
    Co-Authors: Gregory W Muth, L Chen, Anne B Kosek, Scott A Strobel
    Abstract:

    A universally conserved adenosine, A2451, within the ribosomal Peptidyl Transferase center has been proposed to act as a general acid-base catalyst during peptide bond formation. Evidence in support of this proposal came from pH-dependent dimethylsulfate (DMS) modification within Escherichia coli ribosomes. A2451 displayed reactivity consistent with an apparent acidity constant (pKa) near neutrality, though pH-dependent structural flexibility could not be rigorously excluded as an explanation for the enhanced reactivity at high pH. Here we present three independent lines of evidence in support of the alternative interpretation. First, A2451 in ribosomes from the archaebacteria Haloarcula marismortui displays an inverted pH profile that is inconsistent with proton-mediated base protection. Second, in ribosomes from the yeast Saccharomyces cerevisiae, C2452 rather than A2451 is modified in a pH-dependent manner. Third, within E. coli ribosomes, the position of A2451 modification (N1 or N3 imino group) was analyzed by testing for a Dimroth rearrangement of the N1-methylated base. The data are more consistent with DMS modification of the A2451 N1, a functional group that, according to the 50S ribosomal crystal structure, is solvent inaccessible without structural rearrangement. It therefore appears that pH-dependent DMS modification of A2451 does not provide evidence either for or against a general acid-base mechanism of protein synthesis. Instead the data suggest that there is pH-dependent conformational flexibility within the Peptidyl Transferase center, the exact nature and physiological relevance of which is not known.