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Davidovich, Chen

Targeting Functional Centers of the Ribosome (Gebundene Ausgabe)


Reihe: Springer Theses

Springer-Verlag GmbH, Februar 2011


74 S. - Sprache: Englisch - 24 farbige Abbildungen, 2 schwarz-weiße Tabellen - 243x161x14 mm Book

ISBN: 3642169309 EAN: 9783642169304

This thesis describes research into the mode of function, inhibition, and evolution of the ribosomal catalytic center, the Peptidyl Transferase Center (PTC)--research that has already led to attempts at improving PTC antibiotics. The PhD candidate carried out two parallel studies. One using a combination of X-ray crystallography, biochemistry, molecular biology, and theoretical studies to obtain crystal structures of ribosomal particles with antibiotics that target the PTC, revealing the modes of action, resistance, cross-resistance and discrimination between ribosomes of eubacterial pathogens and eukaryotic hosts. In the second parallel study, the candidate synthesized a ribosomal substructure--one that may represent the minimal entity capable of catalyzing peptide bond formation--shedding light on the origin of the ribosome itself.


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Produktdetails

Nominated by the Weizmann Institute, Israel for a Springer Theses PrizeThis research has already led to attempts to improve PTC antibioticsBased on these findings further research into designing a functional construct mimicking a functional proto-ribosome has been initiated

Inhaltsverzeichnis

1. Introduction  1.1 Ribosomal antibiotics: function, activity and selectivity           1.2 Pleuromutilins: a class of PTC ribosomal antibiotics for clinical use   1.3 Structural basis for cross-resistance between ribosomal PTC antibiotics          1.4 Oligonucleotides as ribosomal inhibitors and as tools for structural and functional study       1.5 Minimal ribosomal components with PTC structure and function       2. Methods         2.1 Structural study of pleuromutilin antibiotics               2.1.1 Crystallization and data collection2.1.2 Data processing, structure solution and refinement               2.2 Comparative structural analysis to reveal the structural basis for cross-resistance between PTC antibiotics          2.3 Antisense oligonucleotides for targeting functional ribosomal centers             2.3.1 Database construction         2.3.2 In-vitro transcription-translation system for ribosome activity assay            2.3.3 Antisense oligonucleotides nomenclature   2.4 Minimal ribosomal components with PTC structure and function      2.4.1 In vitro RNA transcription 2.4.2 Study of dimerization tendency      2.4.3 Electrophoresis Mobility Shift Assay (EMSA)           2.4.4 Size Exclusion Chromatography (SEC) for the separation between dimer and monomer      2.4.5 Radiolabeling of substrates for Peptidyl Transferase activity assay  2.4.6 Assay for Peptidyl Transferase activity       2.4.7 RNA two-dimensional structure prediction              2.5 Numbering, Sequence Alignment, and Images.           3. Results            3.1 The structures of D50S/Pleuromutilins comlexes        3.2 Structural basis for cross resistance between ribosomal PTC antibiotics          3.3 Oligonucleotides as ribosomal inhibitors and as tools for structural and functional study       3.3.1 Correlation between IC50 and various ODN parameters       3.3.2. Effect of ODN length          3.4 Minimal ribosomal components with PTC structure and function      3.4.1 Construct design    3.4.2 Study of dimerization tendency      4. Discussion     4.1 Pleuromutilins           4.1.1 Induced-fit mechanism for pleuromutilin binding  4.1.2 C14 extension is located in the PTC void.     4.1.3 Pleuromutilins resistance   4.1.4 Pleuromutilins selectivity acquired by remote interactions               4.2 Structural basis for cross resistance between ribosomal PTC antibiotics          4.2.1 Resistance to PTC antibiotics is frequently acquired by mutating remote nucleotides           4.2.2 U2504 at the crossroad of remote mutations networks that hamper binding of PTC antibiotics.4.2.3 Second layer nucleotides    4.2.4 Third Layer Nucleotides    4.2.5 Resistance to various PTC antibiotics mediated by the same nucleotides      4.3 Oligonucleotides as ribosomal inhibitors and as tools for structural and functional studies    4.4 Minimal ribosomal components with PTC structure and function     5. References      6. Figures, tables and plots         


ISBN 3-642-16930-9

ISBN 978-3-642-16930-4

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