Isolation and characterization of ligands of ribosomal peptidyltransferase centre

Abbreviations _______________________________________________________1 1. Introduction_______________________________________________________3 2. Materials and Methods______________________________________________5 2.1 Materials ____________________________________________________________________5 2.1.1 Suppliers_________________________________________________________________5 2.1.2 Bacterial Strains and Plasmids ________________________________________________8 2.1.3 Media, Gel Solutions, Buffers ________________________________________________9 2.1.3.1 Media________________________________________________________________9 2.1.3.2 Gel Solutions and Electrophoresis Buffers ___________________________________9 2.1.3.3 Plasmid DNA Isolation Buffers___________________________________________11 2.1.3.4 Buffers for the RNA Transcription ________________________________________12 2.1.3.5 Buffers for the in vitro Translation System (Watanabe System and poly(Phe) synthesis system)____________________________________________________________________13 2.1.3.6 Buffers for the HPLC Run_______________________________________________15 2.2 Analytic Methods ____________________________________________________________16 2.2.1 Photometric Measurements _________________________________________________16 2.2.1.1 Spectrophotometric Determination of the Amount of DNA or RNA ______________16 2.2.1.2 Determination of Ribosome and Nucleic Acid Concentration ___________________17 2.2.2 Radioactivity Measurements ________________________________________________18 2.2.2.1 Liquid Samples _______________________________________________________18 2.2.2.2 Nitrocellulose Filters (containing single or multiple labels) _____________________18 2.2.2.3 Glass Fiber Filters with 14C/3H, 14C/32P or 14C/3H/32P Triple Labels _______________18 2.2.2.4 Controls for (multiple) Labels ____________________________________________18 2.2.3 Cold Trichloroacetic Acid (TCA) Precipitation Assay_____________________________19 2.2.4 Electrophoresis Techniques _________________________________________________19 2.2.4.1 Agarose Gel Electrophoresis _____________________________________________19 2.2.4.2 Denaturing Urea-Polyacrylamide Gel Electrophoresis _________________________20 2.3 Preparative Methods __________________________________________________________21 2.4 Genetic Methods: Working with DNA ____________________________________________22 2.4.1 Small Scale Preparation of Plasmid DNA (Miniprep) _____________________________22 2.4.2 Large Scale Preparation of Plasmid DNA (Maxiprep) _____________________________23 2.5 Working with RNA: __________________________________________________________24 2.5.1 Run-off Transcription with T7 Polymerase _____________________________________24 2.5.1.1 Transcribed RNAs Used in this Thesis _____________________________________25 2.5.1.2 In vitro Transcription___________________________________________________25 2.5.2 Direct tRNA Charging _____________________________________________________26 2.5.2.1 Charging of Normal tRNA ______________________________________________26 2.5.2.2 Charging and Purification of fMet-tRNAf Met_________________________________27 2.5.3 Purification of Transcriptions via Polyacrylamide Gel Electrophoresis________________28 2.5.4 3’-Labeling of tRNAPhe with [γ- 32P]pCp ______________________________________29 2.5.4.1 The Labeling Reaction__________________________________________________29 2.5.4.2 Purification of 3'-[γ- 32P]tRNAPhe _________________________________________30 2.5.5 RNase T1 Digestion of 3'-[γ- 32P]-tRNAPhe _____________________________________30 2.5.5.1 The T1 Digestion Step__________________________________________________30 2.5.5.2 Image Quant analysis of Gels ____________________________________________31 2.5.6 Reversed-Phase HPLC Separation of RNase T1 Digested 3'-[γ- 32P]-tRNAPhe Fragments _31 2.6 In vitro Systems______________________________________________________________33 2.6.1 Estimation of the Functional Competence of Ribosome Preparations _________________33 2.6.1.1 Poly(U)-dependent Poly(Phe) Synthesis ____________________________________33 2.6.1.2 Determination of the AcPhe-tRNAPhe Binding ______________________________34 2.6.2 Watanabe Assay: site specific binding of tRNA to ribosomes, translocation and puromycin reaction _____________________________________________________________________34 2.6.2.1 First Step: P site binding or Pi complex formation ____________________________36 2.6.2.2 Second Step: A site binding and/or PRE complex formation ____________________36 2.6.2.3 Third Step: Translocation reaction ________________________________________36 2.6.2.4 Fourth Step: puromycin reaction __________________________________________37 3. Results __________________________________________________________39 3.1 Templates Preparation _________________________________________________________40 3.1.1 Plasmid Identification______________________________________________________40 3.1.1.1Primer Design _________________________________________________________40 3.1.1.2Commercial Sequencing _________________________________________________41 3.1.1.3Analysis of Sequencing Result _________________________________________42 3.1.2 Plasmid Porpagation_______________________________________________________44 3.1.3 Digestion of Plasmid with FokI______________________________________________44 3.2 Optimization of Transcription ___________________________________________________46 3.3 Direct Charging ______________________________________________________________48 3.4 3'-[γ−³²P] Labeling____________________________________________________________51 3.5 T1 Digestion ________________________________________________________________53 3.6 HPLC Purification of T1 Digested 3'-labeled tRNAPhe ________________________________54 4. Discussion________________________________________________________58 4.1 The Ribosome is the Protein Synthesis Machine_____________________________________58 4.2 Two Ribosomal Subunits with Different Tasks______________________________________62 4.3 Two Ribosomal Subunits have Different Architechtures ______________________________64 4.4 The Peptidyl-transferase (PTF) Reaction __________________________________________68 4.5 Strategy for Preparation of PTF Ligands___________________________________________70 5. Zusammenfassung/ Summary _______________________________________73 6. References _______________________________________________________75 7. Acknowledgements ________________________________________________8