Structure and requirement of the Spt6 SH2 domain and an in vitro system to test the torpedo model of transcription termination [Elektronische Ressource] / Stefan Dengl

Stefan Dengl

Sujets

• Biologie

Informations

• Publié par: ludwig-maximilians-universitat_munchen
• Publié le: 01 janvier 2009
• Nombre de lectures: 11
• 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


Structure and requirement of the Spt6
SH2 domain and an in vitro system to test
the “torpedo model” of transcription
termination





Stefan Dengl
aus
München

2009
Erklärung II
Erklärung
Diese Dissertation wurde im Sinne von §13 Abs. 3 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, am





_______________________________
Stefan Dengl





Dissertation eingereicht am

1. Gutachter: Prof. Dr. Patrick Cramer
2. Gutachter: Prof. Dr. Dietmar Martin

Mündliche Prüfung am 25. März 2009
Acknowledgements III
Acknowledgements

First of all I would like to thank my supervisor Prof. Dr. Patrick Cramer for giving me the
opportunity to work on challenging projects in a highly interdisciplinary manner. The freedom
to realize own ideas in combination with your continuous advice and motivation is invaluable
for the enthusiasm of a young scientist.

I want to thank my collaborators Andreas Mayer, Mai Sun, Kristin Leike, Matthias Siebert and
Johannes Soeding for their work and the discussions, but also for a lot fun and reflief from
drawbacks in the projects. It was really a pleasure to work with you guys!

Very special thanks go out to Claus Kuhn and Stephan Jellbauer, not only for teaching me
crystallography and yeast work, respectively, but most important for your friendship in good
and in hard times. The legendary "coffee-at-11.30" was often enough an escape from
scientific misery and sometimes gave birth to new ideas. I will never forget the time we spent
together, also outside of the lab.

I wish to thank Elisabeth, Jasmin, Gerke and Florian for their help with elongation complexes.
Laurent, Ale and Dirk for support concerning crystallography. Ania for advice on the ChIP-on-
chip protocol. Claudia and Stefan Benkert for help with everything, without you the lab would
be a battlefield. Many thanks to Heidi Feldmann for help with yeast work and for not killing
me for some of my jokes. Thank you Gregor for illuminating me with your fluorescent
expertise. I want to thank the former members of the lab, Hubert, Karim, Michela, Sabine,
Sonja, Toni, and Tomislav for a lot of support especially in the beginnig. I want to thank you,
Erika, for being an accomplice in "chromatin-related" issues that were otherwise quite exotic
in the lab.
Of course I want to thank everybody else in the Cramer group for help, media, buffers, DNA
and protein standards, gels, coffee, cake, meaningful discussions and silly conversations.

Von ganzem Herzen danke ich meinen Eltern und meiner Oma Elisabeth. Ohne eure
moralische, aber auch finanzielle Unterstützung wäre das Studium und so viel mehr
undenkbar gewesen.

Dir Moni danke ich für das schöne letzte Jahr und dass du mir immer gute Laune verordnet
hast auch wenn die Batterie mal leer war. Summary IV
Summary

During the biogenesis of messenger RNA, RNA polymerase II (RNAP II, Pol II) associates
with numerous proteins and multiprotein complexes. These factors regulate the correct
progression of the transcription cycle, which can be divided into three major phases:
initiation, elongation and termination. This thesis describes the characterization of two
important factors involved in two different phases of the transcription cycle in a highly
interdisciplinary approach. Spt6 is an essential, modular protein involved in transcription
elongation. It was characterized as a histone chaperone that binds to histones and
assembles nucleosomes onto DNA after the passage of Pol II. In addition, it is linked to
processes like splicing, mRNA processing and export, and histone modification. This
functional versatility makes it a central player in the elongation process. The first part of this
work shows the high resolution structure of the C-terminal SH2 domain of Spt6. The domain
was shown previously to interact with the C-terminal domain (CTD) of Pol II, phosphorylated
at Ser residues. Thus it links Spt6 functions directly to the transcription machinery. The 2
domain has an unconventional binding specifity in contrast to the numerous SH2 domains
involved in cellular signaling pathways. It binds phosphoserine instead of phosphotyrosine. In
addition, it is the only SH2 domain encoded in the yeast genome, opposing a multitude of
these domains in the genomes of higher eukaryotes. The X-ray structure gives insight into
the peculiarities of this domain, with implications on its substrate specificity and molecular
evolution. In this light, a model for the interaction with the CTD, as well as a deep analysis of
the evolutionary relationship to other SH2 domains is presented. Microarray gene expression
analysis shows the impact of a deletion of the SH2 domain on the transcription of the yeast
genome. A genome wide localization map of Spt6, obtained by ChIP-on-chip experiments, is
presented and compared to the localization of Pol II.
The nuclear exoribonuclease complex Rat1/Rai1 plays a role in transcription termination.
Two models explain the events at the end of a protein coding gene. The so-called „torpedo
model“ states that Rat1/Rai1 processively degrades 3’ nascent RNA that is still attached to
elongating Pol II after the mRNA product is cleaved by cleavage/polyadenylation factors.
Upon contact with Pol II, Rat1/Rai1 is thought to disrupt the elongation complex and
terminate transcription. There is however also data supporting the "allosteric model" that
predicts the recruitment of a termination factor or the dissociation of an anti-termination
factor. The second part of this thesis describes the establishment of a highly defined
biochemical assay to test the "torpedo model" in vitro. A protocol for the expression and
purification of the recombinant and active exonuclease complex and an additional interacting
protein, Rtt103, was established. In combination with an improved in vitro elongation assay it
is shown that Rat1 is not the dedicated termination factor that is predicted by the torpedo
model. Publications V
Publications
Parts of this work have been published or are in the process of publication:


Dengl S., Mayer, A., Sun, M., Cramer, P. (2009). Structure and widespread requirement of
the Spt6 SH2 domain, the only SH2 domain in yeast. Journal of molecular biology, in
revision.


Dengl S., Cramer P. (2009). The torpedo nuclease Rat1 is insufficient to terminate RNA
polymerase II in vitro. The Journal of biological chemistry, in revision.


Sydow, J. F. , Brueckner, F., Dengl, S., Cramer P. (2009). Molecular basis of RNA
polymerase II fidelity: mismatch specificity and RNA fraying. Manuscript in preparation.


Cramer P, Armache KJ, Baumli S, Benkert S, Brueckner F, Buchen C, Damsma GE, Dengl
S, Geiger SR, Jasiak AJ, Jawhari A, Jennebach S, Kamenski T, Kettenberger H, Kuhn CD,
Lehmann E, Leike K, Sydow JF, Vannini A (2008) Structure of eukaryotic RNA polymerases.
Annual review of biophysics 37: 337-352






Table of contents VI
Table of contents

Erklärung ....................................................................................................................II
Ehrenwörtliche Versicherung ...................................................................................II
Acknowledgements ..................................................................................................III
Summary ...................................................................................................................IV
Publications ...............................................................................................................V
Table of contents .....................................................................................................VI
1 General introduction .............................................................................................1
1.1 Structure of RNA polymerase II and the elongation complex......................................1
1.2 The C-terminal domain (CTD) of Pol II subunit Rpb1....................................................3
1.3 The mRNA transcription cycle........................................................................................5
1.3.1 Transcription elongation ...................................... .......................................................6
1.3.2 Transcription termination ............................................................................................9
2 Materials and common methods .........................................................................12
2.1 Materials..........................................................................................................................12
2.