Molecular Basis of Rrn3-regulated RNA Polymerase I Initiation [Elektronische Ressource] / Claudia Blattner. Betreuer: Patrick Cramer

Claudia Blattner

Sujets

• Biologie

Informations

• Publié par: ludwig-maximilians-universitat_munchen
• Publié le: 01 janvier 2011
• Nombre de lectures: 16
• Langue: English
• Poids de l'ouvrage: 3 Mo

Extrait

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




Molecular Basis of Rrn3-regulated RNA
Polymerase I Initiation




Claudia Blattner
aus
Lörrach


2011


Erklärung
Diese Dissertation wurde im Sinne von §13 Abs. 3 bzw. 4 der Promotionsordnung vom 29.
Januar 1998 (in der Fassung der sechsten Änderungssatzung vom 16. August 2010) von
Herrn Prof. Dr. Patrick Cramer betreut.

Ehrenwörtliche Versicherung
Diese Dissertation wurde selbstständig, ohne unerlaubte Hilfe erarbeitet.
München, am 14.10.2011



Claudia Blattner






Dissertation eingereicht am 21.10.2011
1. Gutachter Prof. Patrick Cramer
2. Gutachter Prof. Roland Beckmann
Mündliche Prüfung am 05.12.2011


1

Acknowledgements
First of all, I would like to thank Patrick who has been a great supervisor and mentor over
the last years. Thank you for your constant support and advice, and for sharing your
enthusiasm about science. I am very grateful for the opportunity, to do my PhD in this
fantastic lab, and to experience this very productive setting but also social atmosphere.
I want to extend this acknowledgement not only to the lab, but also to the whole scientific
environment I had the chance to work in. This includes the entire Gene Center, with its
great and collaborative atmosphere, and extends to the MPI, Martinsried, and the
research groups and people involved in the IMPRS-ls program, which provided a great
framework for doing research but also getting insight into a broader spectrum of life
sciences and to stay in contact with other researchers. In this context, many thanks to
Maxi and Hans-Jörg, who coordinated all the workshops and other IMPRS activities I could
participate in.
I also got a lot of advice and help from people in the lab. I want to thank Alan for the help
and training in crystallography, for spending countless hours at the synchrotron with me,
explaining, but also having great conversations and drinking whiskey.
Many thanks to Rieke for exchanging our experiences and thoughts throughout our PhD
time, for being a great and fun roommate at all the retreats, and conferences. Thanks to
Elmar for interesting conversations and your enthusiasm about science, Tobi, for
explaining and helping most patiently with so many bioinformatic programs.
Thank you Jenne, for a lot of help in the lab right from the start until today with various
issues, and for your friendship. Many thanks to Jasmin, for endless conversations, for your
general advice on labwork and especially for being a great friend.
Thanks to Kerstin, Sarah and Fuensanta for enjoyable hikes together, parties, and
christmas-cookie sessions.
I also would like to thank Claudia for keeping the lab running and being the source of the
good atmosphere. Thanks to Stefan Benkert, not only for the yeast fermentation, but also
for help with a lot of technical issues.
Many thanks to all the others in the lab, who contributed to the great working
atmosphere, for a great time not only in the lab, but also apart from working hours, during
retreats, beer garden visits and our annual “lab-wiesn”. Also many thanks to my student
2

Sandra, who was a great help in starting the core factor project, I really appreciate your
interest and excitement about this work.
I thank Thomas Fröhlich for frequent and very helpful masspec protein identification.
Further, I would like to acknowledge all the people contributing to the experiments, that
made this work successful. This includes my collaboration partners Kristina Lorenzen from
the lab of Albert Heck at the University of Utrecht, Franz Herzog from the lab of Ruedi
Aebersold at the ETH, Zürich, Gregor Witte from Karl-Peter Hopfner’s lab at the Gene
Center, and Andreas Mayer from our lab.
Many thanks also to the people from the Beckmann lab, Otto Berninghausen, Charlotte
Ungewickell and Thomas Becker who helped me with EM data collection and processing,
and patiently answered all my questions, and, of course, Anselm for his help.
Finally, I want to thank my parents, who aroused my interest in science and supported and
motivated me to make my way, also when things were not easy. And thank you Niklas, for
your patience, your support and understanding.


3

Summary
Eukaryotic nuclear transcription is carried out by three different Polymerases (Pol), Pol I,
Pol II and Pol III. Among these, Pol I is dedicated to transcription of the rRNA, which is the
first step of ribosome biogenesis, and cell growth is regulated during Pol I transcription
initiation by the conserved factor Rrn3/TIF-IA in yeast/human. A wealth of structural
information is available on Pol II and its general transcription factors (GTFs). Recently, also
the architectures of Pol I and Pol III have been described by electron microscopy and the
additional subunits that are specific to Pol I and Pol III have been identified as orthologs of
the Pol II transcription factors TFIIF and TFIIE. Nevertheless, we still lack information about
the architecture of the Pol I initiation complex and structural data is missing explaining the
regulation of Pol I initiation mediated by its central transcription initiation factor Rrn3.
The Rrn3 structure solved in this study reveals a unique HEAT repeat fold and indicates
dimerization of Rrn3 in solution. However, the Rrn3-dimer is disrupted upon Pol I binding.
The Rrn3 structure further displays a surface serine patch. Phosphorylation of this patch
represses human Pol I transcription (Mayer et al, 2005; Mayer et al, 2004), and a phospho-
mimetic patch mutation prevents Rrn3 binding to Pol I in vitro, and reduces S. cerevisiae
cell growth and Pol I gene occupancy in vivo. This demonstrates a conserved regulation
mechanism of the Pol I-Rrn3 interaction. Crosslinking indicates that Rrn3 does not only
interact with Pol I subunits A43/14, but the interface further extends past the RNA exit
tunnel and dock domain to AC40/19. The corresponding region of Pol II binds the
Mediator head (Soutourina et al., 2011) that co-operates with TFIIB (Baek et al, 2006).
Consistent with this, the Rrn3 binding partner, core factor subunit Rrn7, is predicted to be
a TFIIB homologue.
Taken together, our results provide the molecular basis of Rrn3-regulated Pol I initiation
and cell growth and indicate a universally conserved architecture of eukaryotic
transcription initiation complexes.

4

Publication
Blattner C, Jennebach S, Herzog F, Mayer A, Cheung AC, Witte G, Lorenzen K, Hopfner K-P,
Heck AJR, Aebersold R, Cramer P (2011). Molecular basis of Rrn3-regulated RNA
polymerase I initiation and cell growth. Genes Dev 25(19): 2093-2105.

Contributions
The many experiments performed and results presented in this study were achieved with
the help, advice and collaboration of several specialized researchers, whose contributions
are listed below in detail.

Claudia Blattner carried out all experiments apart from the ones listed below and
determined the X-ray structure of Rrn3 with help and advice from Alan Cheung.
SAXS analysis of the Rrn3 dimer was carried out by Gregor Witte.
Native MS was carried out by Kristina Lorenzen.
Protein crosslinking-MS analysis was carried out by Stefan Jennebach and Franz Herzog.
ChIP analysis was done by Andreas Mayer.
5

Contents
Erklärung..................................................................................................................................... 1
Ehrenwörtliche Versicherung ..... 1
Acknowledgements .................................................................................................................... 2
Summary ..................................................................................................................................... 4
Publication .. 5
Contributions .............................. 5
Contents ..................................................................................................................................... 6
1 Introduction ............................. 9
1.1 Eukaryotic Transcription Systems ................................................................................ 9
1.1.1 General Transcription Factors (GTFs) ...... 11
1.1.2 Structural studies on eukaryotic polymerase systems............ 12
1.2 rDNA transcription in the context of ribosome biogenesis and function .................. 14
1.2.1 Ribosome composition and biogenesis ................................................................... 14
1.2.2 rRNA production ...................................... 16
1.3 RNA Polymerase I transcription initiation complex ................................................... 17
1.3.1 Organization of the rRNA genes .............................................. 17
1.3.2 Initiation complex formation in mammals .............................. 17
1.3.3 Initiation complex formation in yeast ..................................... 18
1.4 Regul