Site specific phosphorylation of yeast RNA polymerase I [Elektronische Ressource] / vorgelegt von Jochen Gerber

Jgerber

Sujets

• Biologie

Informations

• Publié par: universitat_regensburg
• Publié le: 01 janvier 2008
• Nombre de lectures: 21
• Langue: Deutsch
• Poids de l'ouvrage: 9 Mo

Extrait

Site-specific phosphorylation of yeast RNA polymerase I











DISSERTATION ZUR ERLANGUNG DES DOKTORGRADES DER
NATURWISSENSCHAFTEN (DR. RER. NAT.) DER NATURWISSENSCHAFTLICHEN
FAKULTÄT III – BIOLOGIE UND VORKLINISCHE MEDIZIN DER UNIVERSITÄT
REGENSBURG





vorgelegt von
Jochen Gerber aus Frohnhofen
02/2008





















Promotionsgesuch eingereicht am: 13.02.2008

Die Arbeit wurde angeleitet von: Herbert Tschochner


Prüfungsausschuss:

Vorsitzender: Prof. Dr. Gernot Längst
1. Gutachter: Prof. Dr. Herbert Tschochner
2. Gutachter: Prof. Dr. Michael Thomm
3. Prüfer: Prof. Dr. Reinhard Sterner



Tag der mündlichen Prüfung: 28.04.2008



Die vorliegende Arbeit wurde unter Anleitung von Prof. Dr. Herbert Tschochner am
Biochemie-Zentrum (BZH) der Universität Heidelberg sowie am Institut für Biochemie,
Genetik und Mikrobiologie der Universität Regensburg angefertigt.






















Ich erkläre hiermit, dass ich diese Dissertation selbst verfasst und keine anderen als die
angegebenen Quellen und Hilfsmittel verwendet habe. Diese Arbeit war bisher noch nicht
Bestandteil eines Prüfungsverfahrens; andere Promotionsversuche wurden nicht
unternommen.


Regensburg, 24.04.2008



Table of contents

1 INTRODUCTION ............................................................................................. 1
1.1 Cell growth and ribosome biogenesis.................................................................................................... 1
1.2 The RNA Polymerase I transcription system of Saccharomyces cerevisiae ....................................... 3
1.2.1 Cellular localization, template and promoter of Pol I........................................................................ 3
1.2.1.1 The nucleolus ............................................................................................................................. 3
1.2.1.2 The ribosomal DNA genes ......................................................................................................... 4
1.2.1.3 The rDNA promoter and Pol I transcription factors................................................................... 6
1.2.2 Pol I is one of three conserved nuclear multisubunit RNA polymerases........................................... 7
1.2.2.1 The nuclear RNA polymerases and their functions in the cellular transcription apparatus........ 7
1.2.2.2 Composition of yeast Pol I 9
1.2.2.3 The structure of the yeast Pol I complex resembles the general architecture of multisubunit
RNA polymerases..................................................................................................................... 10
1.2.3 Pol I subunits ................................................................................................................................... 13
1.2.3.1 A190 and A135 – the two largest subunits............................................................................... 13
1.2.3.2 AC40 and AC19 – the α-like subunits shared by Pol I and Pol III........................................... 14
1.2.3.3 ABC27, ABC23, ABC14.5, ABC10 α and ABC10 β – the common subunits .......................... 15
1.2.3.4 A43 and A14 – the ‘stalk’ subunits .......................................................................................... 16
1.2.3.5 A12.2 – a TFIIS-like subunit.................................................................................................... 18
1.2.3.6 A49 and A34.5 – Pol I specific subunits without counterparts in other RNA polymerases ..... 20
1.2.4 The life and death of Pol I ............................................................................................................... 21
1.2.4.1 Expression of the Pol I subunits ............................................................................................... 21
1.2.4.2 Assembly and nuclear import of the complex .......................................................................... 22
1.2.4.3 The Pol I transcription cycle..................................................................................................... 23
1.2.4.4 Degradation .............................................................................................................................. 25
1.2.5 Pol I phosphorylation....................................................................................................................... 25
1.2.5.1 Pol I is a phosphoprotein complex ........................................................................................... 25
1.2.5.2 Kinases and phosphatases......................................................................................................... 28
1.3 Identification of phosphorylation sites using mass spectrometry..................................................... 30
1.4 Objective................................................................................................................................................ 32
2 RESULTS ...................................................................................................... 35
2.1 Pol I purification.... 35
2.2 Pol I phosphorylation sites................................................................................................................... 37
2.2.1 Chemical derivatization of phosphopeptides – establishing the method ......................................... 37
2.2.2 Identification of Pol I phosphorylation sites.................................................................................... 41
2.2.3 Localization of the phosphorylation sites in the Pol I complex....................................................... 45
2.3 Mutants of the Pol I phosphosites........................................................................................................ 51
2.3.1 Creating the mutants........................................................................................................................ 51
2.3.2 In vivo analyses of the Pol I phosphorylation site mutants.............................................................. 53
2.4 The A12.2 paradox – A lethal mutation of a non-essential protein .................................................. 56
3 DISCUSSION ................................................................................................ 59
3.1 Pol I purification................................................................................................................................... 59
3.1.1 The new yeast Pol I purification method......................................................................................... 59
3.1.2 Co-purifying proteins and Pol I interaction partners ....................................................................... 60
Table of contents
3.2 Identification of Pol I phosphorylation sites....................................................................................... 60
3.2.1 17 Pol I phosphorylation sites.......................................................................................................... 60
3.2.2 The total Pol I preparation used for phosphosite identification is a mixture of different Pol I
populations ...................................................................................................................................... 62
3.2.3 A similar motif in A190 and A34.5 ................................................................................................. 63
3.3 Possible roles of phosphorylation at the identified sites based on the localization in the Pol I
homology model and mutant phenotypes ........................................................................................... 64
3.3.1 All analyzed Pol I phosphorylations are non-essential post-translational modifications - general
considerations and possible functions.............................................................................................. 64
3.3.2 A190 S354 and A190 S1636 on the polymerase clamp.................................................................. 65
3.3.3 A190 S936/941 in the funnel........................................................................................................... 66
3.3.4 A190 S685 at the backside of the Pore1-domain............................................................................. 67
3.3.5 A43 S220 on the outermost part of the stalk and other sites in the A43 OB-domain ...................... 69
3.3.6 ABC23 S102 in the A190-ABC23-A43 interface ........................................................................... 71
3.4 A mutation in the conserved TFIIS-like motif of the non-essential A12.2 is lethal......................... 73
3.5 Outlook .................................................................................................................................................. 74
4 MATERIALS & METHODS .......................................................................