Structure of the complete RNA polymerase II elongation complex and its interaction with the elongation factor TFIIS [Elektronische Ressource] / Hubert Kettenberger

Kettenberger, Eugène Hubert

Sujets

• Biologie

Informations

• Publié par: ludwig-maximilians-universitat_munchen
• Publié le: 01 janvier 2005
• Nombre de lectures: 5
• Langue: English
• Poids de l'ouvrage: 5 Mo

Extrait

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













Structure of the Complete RNA Polymerase II
Elongation Complex and its Interaction
with the Elongation Factor TFIIS














Hubert Kettenberger
aus Burghausen

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 18. Februar 2005



Hubert Kettenberger
















Dissertation eingereicht am 25.Februar 2005



1. Gutachter: Prof. Dr. Patrick Cramer

2. Gutachter: Prof. Dr. Karl-Peter Hopfner

Mündliche Prüfung am 27. April 2005
ACKNOWLEDGEMENTS

This work is the result of a very enjoyable period in the Gene Centre in the group of Prof.
Patrick Cramer. I am most grateful to Patrick for introducing me to the art and science of
crystallography and for giving me a challenging and most exciting project to work on in an
excellent environment. Patrick’s cheerful and encouraging attitude made working in his group a
very pleasant experience.

Substantial parts of this work were made possible by work of Karim Armache. He pioneered in
establishing key techniques in the young group, and his structure of the complete RNA
polymerase II was indispensible for solving larger structures such as those in my thesis.

Moreover, I am grateful for both scientific and social support from all the members of the
Cramer lab. It was a precious fortune to work in a group of people who readily collaborate, share
ideas and are great folks to work and hang out with. Special thanks to Claudia Buchen for getting
me started with biochemical labwork and to Sabine Höppner, Sonja Baumli, Erika Vojnic, Karim
Armache and Tomislav Kamenski for their friendship. Many thanks to Florian Brückner for
continuing on the Pol II project.

I would also like to thank Prof. Michael Famulok and his group for very good collaboration, as
well as Prof. Karl-Peter Hopfner for help with computing issues and for being in my thesis
committee, together with Prof. Dirk Eick.

I am most indebted to Felix Hartlepp for being a central part of my life. He was the one who
endured my „biochemical questions“ at almost any time of the day.

My parents are the ones who deserve my deepest gratefullness. They supported me mentally,
materially and cake-wise during the past nine years of my studies and thus contributed vitally to
this episode of my life.
MEINEN ELTERN T ABLE OF CONTENTS
SUMMARY

PUBLICATIONS

PART 1: INTRODUCTION
1 Flow of genetic information .................................................................................................... 1
2 Discovery of RNA polymerases.............................................................................................. 2
3 Three classes of cellular RNA polymerases........................................................................... 2
4 Mechanism of DNA-dependent RNA polymerases ............................................................ 3
5 Transcription factors regulate RNA polymerases................................................................. 4
6 Composition of multi-subunit polymerases 4
7 Structure determination of RNA polymerases...................................................................... 6
7.1 Single-subunit RNA polymerases............................................................................................ 6
7.2 Multi-subunit RNA polymerases............................................................................................. 9
8 Structure of Pol II and comparison with other multi-subunit RNA polymerases......... 14
8.1 Core Pol II and the subcomplex Rpb4/Rpb7..................................................................... 17
9 The C-terminal domain of Pol II .......................................................................................... 19
10 The eukaryotic transcription cycle ........................................................................................ 20
10.1 Preinitiation and initiation ...................................................................................................... 20
10.2 Promoter clearance and early elongation ............................................................................. 21
10.3 Elongation and the nucleotide addition cycle...................................................................... 22
10.4 Termination and mRNA processing in eukaryotes ............................................................ 23
10.5 Pol II recycling ......................................................................................................................... 24
11 Elongation factors that regulate Pol II activity ...................................................................24
12 Discovery and function of TFIIS.......................................................................................... 27
13 Transcription in vitro ................................................................................................................30
14 Structure determination of multi-subunit complexes.........................................................30
15 Open questions and scope of this work............................................................................... 33 T ABLE OF CONTENTS



PART 2: RESULTS AND DISCUSSION
16 RNA polymerase II-nucleic acid complexes........................................................................ 34
16.1 Assembly of complete Pol II-bubble-RNA complexes ..................................................... 34
16.2 Characterisation of Pol II-DNA/RNA complexes ............................................................ 37
16.3 Crystallisation of Pol II-nucleic acid complexes ................................................................. 39
16.4 Characterisation of complete Pol II-DNA/RNA cocrystals ............................................ 43
16.5 Data collection ......................................................................................................................... 44
16.6 Phasing and refinement ..........................................................................................................47
16.7 Overview of nucleic acid structure........................................................................................ 50
16.8 DNA unwinding ...................................................................................................................... 53
16.9 RNA displacement and exit ................................................................................................... 55
16.10 Initiation-elongation transition .............................................................................................. 57
16.11 NTP binding, selection, and incorporation ......................................................................... 58
16.12 Comparison with a single-subunit RNA polymerase ......................................................... 62
17 The RNA polymerase II-TFIIS complex............................................................................. 64
17.1 Purification of TFIIS............................................................................................................... 64
17.2 Structural analysis..................................................................................................................... 66
17.3 Induced folding of TFIIS....................................................................................................... 68
17.4 TFIIS extends from a polymerase jaw to the active site.................................................... 72
17.5 TFIIS opens a crevice in the funnel...................................................................................... 73
17.6 TFIIS domain III inserts into the pore ................................................................................ 73
17.7 The TFIIS acidic hairpin complements the polymerase active site.................................. 74
17.8 RNA cleavage........................................................................................................................... 76
17.9 Switching between polymerisation and cleavage................................................................. 77
17.10 Proofreading............................................................................................................................. 77
17.11 Pore restriction and RNA backtracking 78
17.12 TFIIS remodels the polymerase active centre ..................................................................... 78
17.13 TFIIS realigns RNA ................................................................................................................ 80
17.14 Functional conform