Molecular basis of RNA polymerase III transcription repression by Maf1 & Structure of human mitochondrial RNA polymerase [Elektronische Ressource] / Eva Rieke Ringel. Betreuer: Patrick Cramer

Eva Rieke Ringel

Sujets

• Biologie

Informations

• Publié par: ludwig-maximilians-universitat_munchen
• Publié le: 01 janvier 2011
• Nombre de lectures: 26
• Langue: English
• Poids de l'ouvrage: 14 Mo

Extrait

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








Molecular basis of RNA polymerase III transcription
repression by Maf1

&

Structure of human mitochondrial RNA polymerase









Eva Rieke Ringel
aus
Essen







2011


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








Molecular basis of RNA polymerase III transcription
repression by Maf1

&

Structure of human mitochondrial RNA polymerase






















Eva Rieke Ringel
aus
Essen







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 selbständig, ohne unerlaubte Hilfe erarbeitet.


München, .....................................





....................................................................
Eva Rieke Ringel
















Dissertation eingereicht am 26.05.2011

1. Gutachter Prof. Dr. Patrick Cramer

2. Gutachter Prof. Dr. Dietmar Martin

Mündliche Prüfung am 26.07.2011


Acknowledgements


Life-science is like teamsports. If you want to play in a high league, you need to have good players and, even
more importantly, a strong and diehard team effort. Without good passes from your teammates you would never
score a goal and without the right tactics, training input and motivation from your coach, there would be nothing
to win. I am very grateful that I was part of such a successful and inspiring squad, the Cramer lab team.

I want to thank Patrick, the coach, not only for letting me be part of this team but also for his leadership. You
gave me at the right time a lot of freedom to decide over my daily labwork and provided helpful feedback and
project plans, when it was required. You trusted in me and my capabilities, like representing the Pol III team on a
conference in the US. You also motivated me to start the risky, challenging but also extremely exciting “mito
Pol” project, and in the end it worked out and was worse it!

Special thanks go to Dmitry, who initiated the mitoRNAP match! I learned so much about single subunit
polymerases from you! Only your enthusiasm about these tiny initial crystals, and your staying power, enabled
the success of this project. Thanks for sharing many ideas for experiments with me and explaining in long emails
good biochemistry. And yes, the next time you visit the Genecenter, there will be a cold Bavarian beer in the
fridge again.

I would also like to thank my teammates in the Maf1/Pol III match, Anselm Kusser and Alessandro Vannini!
Your passes with plenty of Pol III purifications and cryo EM reconstructions were wonderful and “this time it
worked”. I am very glad that our Pol III team succeeded not only in science but also beyond (and that I got your
famous tiramisu recipe, Ale and your delicious restaurant tip, Anselm).

Many teammates in the lab did not only help with advices, supports and discussions, but also contributed to the
fruitful atmosphere at work. Thank you: Alan, for so much help at the synchrotron, with data processing, and
discussions about crystallography. You are a famous teacher! Christian, for discussions about life beyond
science and for teaching me, a convinced child of the Ruhrpott, the beauty of your home. Claudia (Blattner), for
mastering our PhD times side by side, for sharing uncountable lunch times and for always barely listening and
speaking about all these enjoyments, doubts and thoughts in this time. Claudia (Buchen), for keeping the lab
running and being always a great help for finding everything. Dirk, for providing a lot of expertise in
crystallography and ideas to process the data and build the models even a bit better. Elisabeth, for helping me
with the RNA-extension assays and sharing the great experience in the lab. Elmar, for many useful ideas in the
daily lab work, for your contagious enthusiasm at the bench, and for spending many hours in the lab speaking
about everything under the sun. Jasmin, for sharing her expertise with the bead-based transcription assays and of
course for your “krass” famous Persian meals. Jenne, for discussing with me about soccer and all the other
important things in life and of course for sharing your exceptional theories about the Pol I architecture. Laurent,
for his open-minded interest and challenging questions. Martin, for his advices and help to establish his
transcription assay in the Pol III system. Stefan (Benkert) for fermenting hugh amounts of Pol III. Tobias, for
helping with Äkta-systems also late in the evening, for listening to the latest successes and failures of
experiments, and of course for sharing many delicious coffees.

Also I would like to thank my students Lukas and Alexander.You were more than only substitutes of the team,
but really offered great help in the lab. I learned a lot from teaching you and appreciate your interest in my
projects.

Additionally, many thanks go to Hans-Joerg and Maxi from the IMPRS of the MPI Martinsried. I will profit
from your continuous work to offer students good trainings, suitable workshops, and interesting talk schedules –
a real trainingscamp, so to say.

Auch meinen Eltern, meinem Bruder und meinen Großeltern möchte ich danken. Danke, dass ihr mir die Freiheit
geschaffen habt, zu tun, was ich möchte und mir die Unterstützung gegeben habt, die ich dafür brauche!
Danke Robin, für Dein Verständnis und Deine Hilfe und dass Du mich immer daran erinnerst, was wirklich
wichtig ist!

I
Summary

Topic I
Molecular basis of RNA polymerase III transcription repression by Maf1

RNA polymerase III (RNAP III) is a conserved 17-subunit enzyme that transcribes genes encoding short
untranslated RNAs such as transfer RNAs (tRNAs) and 5S ribosomal RNA (rRNA). These genes are essential
and involved in fundamental processes like protein biogenesis; hence RNAP III activity needs to be tightly
regulated. RNAP III is repressed upon stress and this is regulated by Maf1, a protein conserved from yeast to
humans. Many stress pathways were shown to converge on Maf1 and result in its phosphorylation, followed by
its nuclear import and eventual repression of RNAP III activity. However, the molecular mechanisms of this
repression activity were not known at the beginning of these studies.

This work establishes the mechanism of RNAP III specific transcription repression by Maf1. The
crystal structure of Maf1 was solved. It has a globular fold with surface accessible NLS sequences, which sheds
new light on already published results and explains how stress-induced phopshorylation leads to import of Maf1
into the nucleus. Additionally, cryo EM studies and competition assays show that Maf1 binds RNAP III at its
clamp domain and thereby induces structural rearrangements of RNAP III, which inhibits the interaction with
Brf1, a subunit of the transcription initiation factor TFIIIB. This specifically impairs recruitment of RNAP III to
its promoters and implies that Maf1 is a repressor of transcription initiation. Competition and transcription
assays show that Maf1 also binds RNAP III that is engaged in transcription, leaving RNAP III activity intact but
preventing re-initiation.


Topic II
Structure of human mitochondrial RNA polymerase

The nuclear-encoded human mitochondrial RNAP (mitoRNAP) transcribes the mitochondrial genome, which
encodes rRNA, tRNAs and mRNAs. MitoRNAP is a single subunit (ss) polymerase, related to T7 bacteriophage
and chloroplast polymerases. All share a conserved C-terminal core, whereas the N-terminal parts of mitoRNAP
do not show any homology to other ss RNAPs. Unlike phage RNAPs, which are self-sufficient, human
mitoRNAP needs two essential transcription factors for initiation, TFAM and TFB2M. Both of these factors are
likely to control the major steps of transcription initiation, promoter binding and melting. Thus human
mitoRNAP has evolved a different mechanism for transcription initiation and exhibits a unique transcription
system. Structural studies thus far concentrated on the nuclear enzymes or phage RNAPs, whereas the structure
of mitochondrial RNA polymerase remained