Crystal structures of the complete 12-subunit RNA polymerase II and its subcomplex Rpb4-7, and modeling of RNA polymerases I and III [Elektronische Ressource] / Karim-Jean Armache

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














Crystal structures of the complete 12-subunit
RNA polymerase II and its subcomplex Rpb4/7,
and modeling of RNA polymerases I and III













Karim-Jean Armache
aus Lodz, Polen
2005

Erklärung
Diese Dissertation wurde im Sinne von §13 Abs. 3 bzw. 4 der Promotionsordnung vom 29.
Januar 1998 von Herrn Prof. Dr. Patrick Cramer betreut.





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


München, den 31. März 2005






Karim-Jean Armache



















Dissertation eingereicht am



1. Gutachter: Prof. Dr. Patrick Cramer

2. Gutachter: Prof. Dr. Michael Meisterernst

Mündliche Prüfung am 17-06-2005

Acknowledgements


I would like to thank with a deep sense of gratitude Prof. Patrick Cramer for allowing me to
carry out this research work in his group of macromolecular crystallography at the Gene
Center, University of Munich. The excellent working conditions and atmosphere, his constant
advice and support, were decisive in guiding this work.

Thanks to Hubert Kettenberger for the fruitful collaboration, which extended over last three
years.

My special appreciation goes to Dr. Anton Meinhart who spent long hours on introducing me
to complicated world of symmetry and gave me a lot of help and advice during solving the
structure of Rpb4/7.

I am indebted to Claudia Buchen who introduced me to tricks and shortcuts of the
biochemistry hard-core bench work which made it so much easier to carry out the technical
part of my research.

I am mostly grateful to all the members of the lab especially Tomislav Kamenski, Sabine
Hoeppner, Sonja Baumli and Erika Vojnic for their help, advice and support during various
stages of this work.

I am also greatly thankful to Simone Mitterweger and Elisabeth Lehmann who were
conducting excellent bachelor work in the lab, for all the help I got from them.

Many great thanks to Dr. Heidi Feldmann for scientific discussions and critical comments,
which helped me to understand and solve many molecular biology problems, which I
encountered during my work.

I would like to thank Prof. Karl-Peter Hopfner for all the help I got in crystallography and for
being in my thesis committee.

I would like to thank also Prof. Ralf-Peter Jansen for critical comments on this thesis.

Thanks to my friends Luca Perabo and Karsten Beck who helped me on several occasions
both socially and scientifically, guys thanks for all the great parties we had and also all the
advice I got from you.

Many heartfelt thanks to Anja Fischer who supported me constantly in the last year and made
it much easier to go through difficult times.

My family, Anna and Akram Armache, Jean-Paul Armache and Danuta Talar for being
always with me and supporting me every single moment of my life: Dziekuje wam kochani za
wszystko co dla mnie zrobiliscie, bez was nie byloby tej pracy, bez waszej ciaglej pomocy
przede wszystkim duchowej, ale takze finansowej. Dziekuje Wam !!!
I
Table of contents
Summary................................................................................................................................................ 1
Chapter I: Introduction........................................................................................................................ 2
Macromolecular assemblies................................................................................................................ 3
X-ray crystallography ......................................................................................................................... 4
RNA polymerase II .............................................................................................................. 5
History of Pol II structure determination........................................................................................ 5
General architecture of Pol II and comparison to bacterial RNA polymerase................................ 9
The CTD............... 12
The subcomplex of Rpb4/7............................................................................................................... 13
The mRNA transcription cycle ......................................................................................................... 16
Initiation of transcription..............................................................................................................................17
Chapter II: Architecture of initiation-competent 12-subunit RNA polymerase II ....................... 28
Abstract............................................................................................................................................. 29
Introduction....................................................................................................................................... 30
Materials and methods ...................................................................................................................... 31
Fermentation and purification of 10-subunit Pol II ...................................................................... 31
Expression and purification of full-length Rpb4/7 ....................................................................... 35
Assembly of 12-subunit Pol II...................................................................................................... 36
Crystallization of 12-subunit Pol II .............................................................................................. 37
Results and Discussion...................................................................................................................... 40
Fermentation of 10-subunit Pol II................................................................................................. 40
Purification of 10-subunit Pol II..... 41 h Rpb4/7 43
Assembly of 12-subunit Pol II.... 46
Crystallization of 12-subunit Pol II... 47
X-ray structure determination of 12-subunit Pol II ...................................................................... 50
Overall structure ........................................................................................................................... 51
Rpb7 forms a conserved wedge that restrains the clamp.............................................................. 53
Implications for transcription initiation ........................................................................................ 54
Crystal packing in 12-subunit Pol II facilitates obtaining of higher order complexes ................. 56
Conclusion.................................................................................................................................... 57
Chapter III: Structures of complete RNA polymerase II and its subcomplex Rpb4/7................. 58
Abstract............................................................................................................................................. 59
Introduction....................................................................................................................................... 60
Materials and methods ...................................................................................................................... 61
Design of Rpb4/7 variants ............................................................................................................ 61
Expression and purification of Rpb4/7 variants ........................................................................... 63
Crystallization of Rpb4/7 variants ................................................................................................ 63
Results and discussion ...................................................................................................................... 64
Design of Rpb4/7 variants...... 64
Cloning of Rpb4/7 variants........................................................................................................... 67
Expression, purification, and crystallization of Rpb4/7 variants.................................................. 71
Structure determinations............................................................................................................... 80
II
Rpb4/7 structure ........................................................................................................................... 82
Folding transitions upon Pol II core-Rpb4/7 interaction .............................................................. 82
Specificity of the Pol II core-Rpb4/7 interaction.......................................................................... 86
Importance of the refined 12-subunit Pol II in phasing new structures........................................ 86
Chapter IV: Modeling of