50S Ribosomal Subunit

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Scott W Champney - One of the best experts on this subject based on the ideXlab platform.

  • characteristics of a 50S Ribosomal Subunit precursor particle as a substrate for erme methyltransferase activity and erythromycin binding in staphylococcus aureus
    RNA Biology, 2007
    Co-Authors: Indira Pokkunuri, Scott W Champney
    Abstract:

    Erythromycin is a macrolide antibiotic that inhibits not only mRNA translation but also 50S Ribosomal Subunit assembly in bacterial cells. An important mechanism of erythromycin resistance is the methylation of 23S rRNA by erm methyl transferase enzymes. A model for 50S Ribosomal Subunit formation suggests that the precursor particle which accumulates in erythromycin treated cells is the target for methyl transferase activity. Hybridization experiments identified the presence of 23S rRNA in the 50S precursor particle. The protein content of the 50S precursor particle was analyzed by MALDI-TOF mass spectrophotometry. These studies have identified 23 of 36 50S Ribosomal proteins in the precursor. Methyltransferase assays demonstrated that the 50S precursor particle was a substrate for ermE methyltransferase. Competition experiments indicated that the enzyme could displace erythromycin from the 50S precursor particle and that the methyltransferase had a higher association constant for the precursor particle compared to that of erythromycin. Inhibition experiments showed that macrolide, lincosamide and streptogramin B compounds bound to the precursor particle with similar affinity and inhibited the ermE methyltransferase activity. These studies shed light on the interaction of ermE methyltransferase and erythromycin in this clinically important pathogen.

  • retapamulin inhibition of translation and 50S Ribosomal Subunit formation in staphylococcus aureus cells
    Antimicrobial Agents and Chemotherapy, 2007
    Co-Authors: Scott W Champney, Ward Rodgers
    Abstract:

    Retapamulin inhibited protein biosynthesis and cell viability in methicillin-sensitive and methicillin-resistant Staphylococcus aureus organisms. A specific inhibitory effect on 50S Ribosomal Subunit formation was also found. Pulse-chase labeling experiments confirmed the specific inhibition of 50S Subunit biogenesis. Turnover of 23S rRNA was found, with no effect on 16S rRNA amounts.

  • a 50S Ribosomal Subunit precursor particle is a substrate for the ermc methyltransferase in staphylococcus aureus cells
    Current Microbiology, 2003
    Co-Authors: Scott W Champney, Harold S Chittum, Craig L. Tober
    Abstract:

    Macrolide antibiotics like erythromycin can induce the synthesis of a specific 23S rRNA methyltransferase which confers resistance to cells containing the erm gene. Erythromycin inhibits both protein synthesis and the formation of 50S Subunits in bacterial cells. We have tested the idea that the 50S precursor particle that accumulates in antibiotic-treated Staphylococcus aureus cells is a substrate for the methyltransferase enzyme. Pulse-chase labeling studies were conducted to examine the rates of Ribosomal Subunit formation in control and erythromycin-induced cells. Erythromycin binding to 50S Subunits was examined under the same conditions. The rate of 50S Subunit formation was reduced for up to 30 min after antibiotic addition, and erythromycin binding was substantial at this time. A nuclease protection assay was used to examine the methylation of adenine 2085 in 23S rRNA after induction. A methyl-labeled protected RNA sequence was found to appear in cells 30 min after induction. This protected sequence was found in both 50S Subunits and in a Subunit precursor particle sedimenting at about 30S in sucrose gradients. 23S rRNA isolated from 50S Subunits of cells could be labeled by a ribosome-associated methlytransferase activity, with (3)H-S-adenosylmethionine as a substrate. 50S Subunits were not a substrate for the enzyme, but the 30S gradient region from erythromycin-treated cells contained a substrate for this activity. These findings are consistent with a model that suggests that antibiotic inhibition of 50S formation leads to the accumulation of a precursor whose 23S rRNA becomes methylated by the induced enzyme. The methylated rRNA will preclude erythromycin binding; thus, assembly of the particle and translation become insensitive to the inhibitory effects of the drug.

  • telithromycin inhibition of protein synthesis and 50S Ribosomal Subunit formation in streptococcus pneumoniae cells
    Current Microbiology, 2002
    Co-Authors: Scott W Champney, Jennifer Pelt
    Abstract:

    The new ketolide antibiotic telithromycin (HMR3647) has been examined for inhibitory effects in cells of Streptococcus pneumoniae. The antibiotic caused a proportional decline in cell growth rate and viability with an IC50 of 15 ng/ml. At a concentration of 7.5 ng/ml, protein synthesis in these cells was reduced by 50%. As seen in other organisms, this compound was also a very effective inhibitor of the formation of the 50S Ribosomal Subunit in growing cells. Pulse and chase labeling assays defined the reduced rate of 50S synthesis in antibiotic treated cells. At 7.5 ng/ml the rate was reduced to 50% of the control synthesis rate. An IC50 of 15 ng/ml was found for the effect on this process. 30S Ribosomal Subunit formation was unaffected by the antibiotic. Inhibition of translation and 50S particle formation are equivalent targets for this antibiotic. The effects of telithromycin in S. pneumoniae are compared with those found in Staphylococcus aureus cells.

  • the ketolide antibiotic abt 773 is a specific inhibitor of translation and 50S Ribosomal Subunit formation in streptococcus pneumoniae cells
    Current Microbiology, 2002
    Co-Authors: Scott W Champney, Jennifer Pelt
    Abstract:

    ABT-773 is a new 3-keto macrolide antibiotic that has been shown to be very effective against infections by Gram-positive microorganisms. This work examines its inhibitory effects in cells of Streptococcus pneumoniae. ABT-773 caused a proportional decline in cell growth rates and viability with an IC(50) of 5 ng/ml. Protein synthesis in these cells was reduced by 50% at an antibiotic concentration of 2.5 ng/ml. This compound was also found to be a very effective inhibitor of the formation of the 50S Ribosomal Subunit in growing cells. Pulse and chase labeling assays revealed a reduced rate of 50S synthesis in antibiotic-treated cells. At 2 ng/ml, the rate was reduced to 33% of the control synthesis rate. An IC(50) of 5 ng/ml was found for the effect on this process, indicating an equal effect of the drug on translation and assembly. Synthesis of the 30S Ribosomal Subunit was unaffected by this antibiotic. The effects of ABT-773 in S. pneumoniae are compared with those of the related ketolide antibiotic telithromycin in S. pneumoniae and in Staphylococcus aureus.

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

  • resistance to linezolid caused by modifications at its binding site on the ribosome
    Antimicrobial Agents and Chemotherapy, 2012
    Co-Authors: Katherine S Long, Birte Vester
    Abstract:

    Linezolid is an oxazolidinone antibiotic in clinical use for the treatment of serious infections of resistant Gram-positive bacteria. It inhibits protein synthesis by binding to the peptidyl transferase center on the ribosome. Almost all known resistance mechanisms involve small alterations to the linezolid binding site, so this review will therefore focus on the various changes that can adversely affect drug binding and confer resistance. High-resolution structures of linezolid bound to the 50S Ribosomal Subunit show that it binds in a deep cleft that is surrounded by 23S rRNA nucleotides. Mutation of 23S rRNA has for some time been established as a linezolid resistance mechanism. Although Ribosomal proteins L3 and L4 are located further away from the bound drug, mutations in specific regions of these proteins are increasingly being associated with linezolid resistance. However, very little evidence has been presented to confirm this. Furthermore, recent findings on the Cfr methyltransferase underscore the modification of 23S rRNA as a highly effective and transferable form of linezolid resistance. On a positive note, detailed knowledge of the linezolid binding site has facilitated the design of a new generation of oxazolidinones that show improved properties against the known resistance mechanisms.

  • 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, Lena B Johansson, M. Karlsson, 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.

Thomas A Steitz - One of the best experts on this subject based on the ideXlab platform.

  • initiation factor 2 crystal structure reveals a different domain organization from eukaryotic initiation factor 5b and mechanism among translational gtpases
    Proceedings of the National Academy of Sciences of the United States of America, 2013
    Co-Authors: Daniel Eiler, Jinzhong Lin, Angelita Simonetti, Bruno P Klaholz, Thomas A Steitz
    Abstract:

    The initiation of protein synthesis uses initiation factor 2 (IF2) in prokaryotes and a related protein named eukaryotic initiation factor 5B (eIF5B) in eukaryotes. IF2 is a GTPase that positions the initiator tRNA on the 30S Ribosomal initiation complex and stimulates its assembly to the 50S Ribosomal Subunit to make the 70S ribosome. The 3.1-A resolution X-ray crystal structures of the full-length Thermus thermophilus apo IF2 and its complex with GDP presented here exhibit two different conformations (all of its domains except C2 domain are visible). Unlike all other translational GTPases, IF2 does not have an effecter domain that stably contacts the switch II region of the GTPase domain. The domain organization of IF2 is inconsistent with the “articulated lever” mechanism of communication between the GTPase and initiator tRNA binding domains that has been proposed for eIF5B. Previous cryo-electron microscopy reconstructions, NMR experiments, and this structure show that IF2 transitions from being flexible in solution to an extended conformation when interacting with Ribosomal complexes.

  • a structural view on the mechanism of the ribosome catalyzed peptide bond formation
    Biochimica et Biophysica Acta, 2009
    Co-Authors: Miljan Simonovic, Thomas A Steitz
    Abstract:

    The ribosome is a large ribonucleoprotein particle that translates genetic information encoded in mRNA into specific proteins. Its highly conserved active site, the peptidyl-transferase center (PTC), is located on the large (50S) Ribosomal Subunit and is comprised solely of rRNA, which makes the ribosome the only natural ribozyme with polymerase activity. The last decade witnessed a rapid accumulation of atomic-resolution structural data on both Ribosomal Subunits as well as on the entire ribosome. This has allowed studies on the mechanism of peptide bond formation at a level of detail that surpasses that for the classical protein enzymes. A current understanding of the mechanism of the ribosome-catalyzed peptide bond formation is the focus of this review. Implications on the mechanism of peptide release are discussed as well.

  • crystal structure of the oxazolidinone antibiotic linezolid bound to the 50S Ribosomal Subunit
    Journal of Medicinal Chemistry, 2008
    Co-Authors: Joseph A Ippolito, Thomas A Steitz, Peter B Moore, Zoltan F Kanyo, Deping Wang, Francois Franceschi, Erin M Duffy
    Abstract:

    The oxazolidinone antibacterials target the 50S Subunit of prokaryotic ribosomes. To gain insight into their mechanism of action, the crystal structure of the canonical oxazolidinone, linezolid, has been determined bound to the Haloarcula marismortui 50S Subunit. Linezolid binds the 50S A-site, near the catalytic center, which suggests that inhibition involves competition with incoming A-site substrates. These results provide a structural basis for the discovery of improved oxazolidinones active against emerging drug-resistant clinical strains.

  • structural insights into peptide bond formation
    Proceedings of the National Academy of Sciences of the United States of America, 2002
    Co-Authors: J L Hansen, Peter B Moore, T M Schmeing, Thomas A Steitz
    Abstract:

    The large Ribosomal Subunit catalyzes peptide bond formation and will do so by using small aminoacyl- and peptidyl-RNA fragments of tRNA. We have refined at 3-Å resolution the structures of both A and P site substrate and product analogues, as well as an intermediate analogue, bound to the Haloarcula marismortui 50S Ribosomal Subunit. A P site substrate, CCA-Phe-caproic acid–biotin, binds equally to both sites, but in the presence of sparsomycin binds only to the P site. The CCA portions of these analogues are bound identically by either the A or P loop of the 23S rRNA. Combining the separate P and A site substrate complexes into one model reveals interactions that may occur when both are present simultaneously. The α-NH2 group of an aminoacylated fragment in the A site forms one hydrogen bond with the N3 of A2486 (2451) and may form a second hydrogen bond either with the 2′ OH of the A-76 ribose in the P site or with the 2′ OH of A2486 (2451). These interactions position the α amino group adjacent to the carbonyl carbon of esterified P site substrate in an orientation suitable for a nucleophilic attack.

  • placement of protein and rna structures into a 5 a resolution map of the 50S Ribosomal Subunit
    Nature, 1999
    Co-Authors: Thomas A Steitz, Nenad A, Poul Nisse, J L Hanse, Malcolm Capel, Pete Moore
    Abstract:

    We have calculated at 5.0 A resolution an electron-density map of the large 50S Ribosomal Subunit from the bacterium Haloarcula marismortui by using phases derived from four heavy-atom derivatives, intercrystal density averaging and density-modification procedures. More than 300 base pairs of A-form RNA duplex have been fitted into this map, as have regions of non-A-form duplex, single-stranded segments and tetraloops. The long rods of RNA crisscrossing the Subunit arise from the stacking of short, separate double helices, not all of which are A-form, and in many places proteins crosslink two or more of these rods. The polypeptide exit channel was marked by tungsten cluster compounds bound in one heavy-atom-derivatized crystal. We have determined the structure of the translation-factor-binding centre by fitting the crystal structures of the Ribosomal proteins L6, L11 and L14, the sarcin–ricin loop RNA, and the RNA sequence that binds L11 into the electron density. We can position either elongation factor G or elongation factor Tu complexed with an aminoacylated transfer RNA and GTP onto the factor-binding centre in a manner that is consistent with results from biochemical and electron microscopy studies.

Paola Fucini - One of the best experts on this subject based on the ideXlab platform.

  • the oxazolidinone antibiotics perturb the Ribosomal peptidyl transferase center and effect trna positioning
    Proceedings of the National Academy of Sciences of the United States of America, 2008
    Co-Authors: Daniel N. Wilson, Frank Schluenzen, Joerg Harms, Agata L Starosta, Sean R Connell, Paola Fucini
    Abstract:

    The oxazolidinones represent the first new class of antibiotics to enter into clinical usage within the past 30 years, but their binding site and mechanism of action has not been fully characterized. We have determined the crystal structure of the oxazolidinone linezolid bound to the Deinococcus radiodurans 50S Ribosomal Subunit. Linezolid binds in the A site pocket at the peptidyltransferase center of the ribosome overlapping the aminoacyl moiety of an A-site bound tRNA as well as many clinically important antibiotics. Binding of linezolid stabilizes a distinct conformation of the universally conserved 23S rRNA nucleotide U2585 that would be nonproductive for peptide bond formation. In conjunction with available biochemical data, we present a model whereby oxazolidinones impart their inhibitory effect by perturbing the correct positioning of tRNAs on the ribosome.

  • interaction of era with the 30s Ribosomal Subunit implications for 30s Subunit assembly
    Molecular Cell, 2005
    Co-Authors: Manjuli R Sharma, Paola Fucini, Daniel N. Wilson, C Barat, T M Booth, Masahito Kawazoe, Chie Horitakemoto, Mikako Shirouzu, Shigeyuki Yokoyama, Rajendra K Agrawal
    Abstract:

    Summary Era ( E. coli Ras-like protein) is a highly conserved and essential GTPase in bacteria. It binds to the 16S Ribosomal RNA (rRNA) of the small (30S) Ribosomal Subunit, and its depletion leads to accumulation of an unprocessed precursor of the 16S rRNA. We have obtained a three-dimensional cryo-electron microscopic map of the Thermus thermophilus 30S-Era complex. Era binds in the cleft between the head and platform of the 30S Subunit and locks the Subunit in a conformation that is not favorable for association with the large (50S) Ribosomal Subunit. The RNA binding KH motif present within the C-terminal domain of Era interacts with the conserved nucleotides in the 3′ region of the 16S rRNA. Furthermore, Era makes contact with several assembly elements of the 30S Subunit. These observations suggest a direct involvement of Era in the assembly and maturation of the 30S Subunit.

  • x ray crystallography study on ribosome recycling the mechanism of binding and action of rrf on the 50S Ribosomal Subunit
    The EMBO Journal, 2005
    Co-Authors: Daniel N. Wilson, Frank Schluenzen, Joerg Harms, Renate Albrecht, Takuya Yoshida, Tadayasu Ohkubo, Joerg Buerger, Yuji Kobayashi, Paola Fucini
    Abstract:

    This study presents the crystal structure of domain I of the Escherichia coli ribosome recycling factor (RRF) bound to the Deinococcus radiodurans 50S Subunit. The orientation of RRF is consistent with the position determined on a 70S-RRF complex by cryoelectron microscopy (cryo-EM). Alignment, however, requires a rotation of 7° and a shift of the cryo-EM RRF by a complete turn of an α-helix, redefining the contacts established with Ribosomal components. At 3.3 A resolution, RRF is seen to interact exclusively with Ribosomal elements associated with tRNA binding and/or translocation. Furthermore, these results now provide a high-resolution structural description of the conformational changes that were suspected to occur on the 70S-RRF complex, which has implications for the synergistic action of RRF with elongation factor G (EF-G). Specifically, the tip of the universal bridge element H69 is shifted by 20 A toward h44 of the 30S Subunit, suggesting that RRF primes the interSubunit bridge B2a for the action of EF-G. Collectively, our data enable a model to be proposed for the dual action of EF-G and RRF during ribosome recycling.

  • inhibition of peptide bond formation by pleuromutilins the structure of the 50S Ribosomal Subunit from deinococcus radiodurans in complex with tiamulin
    Molecular Microbiology, 2004
    Co-Authors: Frank Schlunzen, Erez Pyetan, Paola Fucini, Ada Yonath, J Harms
    Abstract:

    Summary Tiamulin, a prominent member of the pleuromutilin class of antibiotics, is a potent inhibitor of protein synthesis in bacteria. Up to now the effect of pleuro- mutilins on the ribosome has not been determined on a molecular level. The 3.5 A structure of the 50S Ribosomal Subunit from Deinococcus radiodurans in complex with tiamulin provides for the first time a detailed picture of its interactions with the 23S rRNA, thus explaining the molecular mechanism of the antimicrobial activity of the pleuromutilin class of antibiotics. Our results show that tiamulin is located within the peptidyl transferase center (PTC) of the 50S Ribosomal Subunit with its tricyclic mutilin core positioned in a tight pocket at the A-tRNA bind- ing site. Also, the extension, which protrudes from its mutilin core, partially overlaps with the P-tRNA binding site. Thereby, tiamulin directly inhibits pep- tide bond formation. Comparison of the tiamulin binding site with other PTC targeting drugs, like chloramphenicol, clindamycin and streptogramins, may facilitate the design of modified or hybridized drugs that extend the applicability of this class of antibiotics.

Katherine S Long - One of the best experts on this subject based on the ideXlab platform.

  • resistance to linezolid caused by modifications at its binding site on the ribosome
    Antimicrobial Agents and Chemotherapy, 2012
    Co-Authors: Katherine S Long, Birte Vester
    Abstract:

    Linezolid is an oxazolidinone antibiotic in clinical use for the treatment of serious infections of resistant Gram-positive bacteria. It inhibits protein synthesis by binding to the peptidyl transferase center on the ribosome. Almost all known resistance mechanisms involve small alterations to the linezolid binding site, so this review will therefore focus on the various changes that can adversely affect drug binding and confer resistance. High-resolution structures of linezolid bound to the 50S Ribosomal Subunit show that it binds in a deep cleft that is surrounded by 23S rRNA nucleotides. Mutation of 23S rRNA has for some time been established as a linezolid resistance mechanism. Although Ribosomal proteins L3 and L4 are located further away from the bound drug, mutations in specific regions of these proteins are increasingly being associated with linezolid resistance. However, very little evidence has been presented to confirm this. Furthermore, recent findings on the Cfr methyltransferase underscore the modification of 23S rRNA as a highly effective and transferable form of linezolid resistance. On a positive note, detailed knowledge of the linezolid binding site has facilitated the design of a new generation of oxazolidinones that show improved properties against the known resistance mechanisms.

  • 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.