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 collection
2.1.2 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
