Single molecule fluorescence studies of the RNA polymerase II elongation complex [Elektronische Ressource] / Joanna Andrecka

Andrecka, Overing Joanna

Informations

• Publié par: ludwig-maximilians-universitat_munchen
• Publié le: 01 janvier 2009
• Nombre de lectures: 11
• Langue: English
• Poids de l'ouvrage: 6 Mo

Extrait

Dissertation zur Erlangung des Doktorgrades der Fakultät für Chemie und Pharmazie
der Ludwig-Maximilians-Universität München



Single molecule fluorescence
studies of the RNA polymerase II
elongation complex








Joanna Andrecka
aus Mys łowice, Polen
2009 Erklärung
Diese Dissertation wurde im Sinne von §13 Abs. 3 der Promotionsordnung vom
29. Januar 1998 von Herrn Prof. Dr. Jens Michaelis betreut.



Ehrenwörtliche Versicherung
Diese Dissertation wurde selbständig und ohne unerlaubte Hilfe erarbeitet.


München, am 22.Juni 2009

Joanna Andrecka





Dissertation eingereicht am 22. Juni 2009
1. Gutachter: Prof. Dr. Jens Michaelis
2. Gutachter: Prof. Dr. Patrick Cramer

Mündliche Prüfung am 17. Juli 2009 Acknowledgements

I would like to thank my supervisor, Prof. Jens Michaelis for creating a highly motivating scientific
environment, lots of freedom but also the pressure he put on me. I am deeply grateful for his support
and understanding of my never-ending trips, which he sometimes called “urlaub - projects”.

Moreover, I would like to thank my collaborator Prof. Patrick Cramer. I have got the unique opportunity
to work in his lab at the time the group was still small - it was an unforgettable experience. I am deeply
thankful to Claudia, Karim, Eli, Flo and Anass for their help.

I would also like to thank all, present and former, members of the AK Bräuchle for creating a very
special atmosphere in our lab. It is difficult to express how great time I had here.
Special thanks to:
Moritz for his “live-advices”;
Robert for making everyday-lab life much easier and for being the most amazing
Bavarian-guy I have ever met;
Adam for all your help, NPS and answering all my stupid questions… Dzi ęki!!!
Peter for vmd script, climbing and night-printing session;
Bärbel for all our „salads” and „coffee breaks” … and of course not only for that;
Iko for encouraging me ☺
All my students, who had to suffer from me - Markus, Julia, Andreas, Thilo… Thanks!

Flo, beside our “collaboration”, thank you for having a real home at Saalburgstrasse 9.

Ania, thanks for bringing me here and that I could always rely on you.

All my friends in Kraków: Agacie- żabie, Kliczowi, Ani Kubasiak, Ani Nikiel, Klemensowi, Markowi,
Wojtkowi, Micha łowi… Dzi ękuj ę Wam Kochani, bez Was nie da łabym rady!!!
oraz Marysi, za drugi dom…

Moim rodzicom, których zaniedba łam…























I wish to dedicate this work to Prof. Zygmunt Wasylewski,
my first supervisor, for whom nothing was impossible Table of contents

SUMMARY
PUBLICATIONS

INTRODUCTION
1 FLUORESCENCE ...................................................................................................................................... 1
1.1 Phenomenon of fluorescence........................................................................................................... 1
1.2 Jab ło ński diagram........................................................................................................................... 2
1.3 Fluorophores................ 4
1.3.1 Natural fluorophores....................................................................................................................... 4
1.3.2 Fluorescent labeling of biomolecules.............................................................................................. 5
1.4 scence Resonance Energy Transfer...................................................................................... 7
1.5 FRET measurements and distance determination........................................................................... 8
1.5.1 Incomplete labeling and averaging effect ....................................................................................... 8
1.5.2 Förster radius.................................................................................................................................. 9
21.5.3 Orientation factor κ ..................................................................................................................... 10
1.6 Single molecule fluorescence ........................................................................................................ 11
1.7 TIRF .............................................................................................................................................. 12
1.8 FRET data used as quantitative distance information .................................................................. 15
2 BAYESIAN DATA ANALYSIS ............................................................................................................... 17
2.1 Bayes’ theorem and marginalization ............................................................................................ 17
2.2 Bayesian approach in data analysis.............................................................................................. 20
2.3 Bayesian analysis in biology and medicine................................................................................... 21
2.4 NPS - bayesian analysis for single molecule FRET data .............................................................. 23
2.4.1 Intuitive description of the analysis method.................................................................................. 23
3 RNA POLYMERASE II ........................................................................................................................... 26
3.1 Transcription................................................................................................................................. 26
3.2 on machinery............................................................................................................... 27
3.3 RNA Pol II transcription cycle ...................................................................................................... 30
3.4 Structure of RNA Pol II elongation complex................................................................................. 33
3.5 Single molecule studies of transcription........................................................................................ 37
3.6 Open questions and scope of this work ......................................................................................... 39

METHODS
4 MOLECULAR BIOLOGY METHODS ................................................................................................. 40
4.1 Single cysteine Rpb4/7 mutants.... 40
4.2 Transformation.............................................................................................................................. 42
4.3 Expression of recombinant protein in E.coli................................................................................. 43
4.4 Purification of the Rpb4/7 subcomplex 44
4.5 Protein labeling............................................................................................................................. 46
4.6 Nucleic acid scaffolds.................................................................................................................... 47
4.7 Preparation of Pol II - artificial bubbles elongation complex ...................................................... 50
4.8 Competition measurements with TFIIB......................................................................................... 50
5 FLUORESCENCE METHODS............................................................................................................... 52
5.1 Anisotropy measurements ............................................................................................................. 52
5.2 Förster radii measurements .......................................................................................................... 52
5.3 Preparation of sample chamber for sp-FRET measurements ....................................................... 53
5.4 Experimental setup for sp-FRET................................................................................................... 55
5.5 Data collection and analysis ......................................................................................................... 58 5.5.1 Camera settings used in single molecule FRET measurements .................................................... 58
5.5.2 Analysis program .......................................................................................................................... 58
5.5.3 Calculation of FRET efficiencies .................................................................................................. 60
6 DATA ANALYSIS METHODS ............................................................................................................... 62
6.1.1 Conventional Method - Triangulation........................................................................................... 62
6.2 NPS................................................................................................................................................ 63
6.2.1 Bayesian parameter estimation applied to NPS...................