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Language(s):
eng - English
Dates:
2003-04
Publication Status:
Accepted / In Press
Pages:
-
Publishing info:
Berlin : Freie Universität
Table of Contents:
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 ________________________________________________83
Rev. Type:
-
Identifiers:
eDoc: 194839
Degree:
Master
