Molekulare Funktion und Regulation des negativen Cofaktors 2, NC2 [Elektronische Ressource] / Elisa Piaia
152
pages
Deutsch
Documents
2005
Obtenez un accès à la bibliothèque pour le consulter en ligne En savoir plus
Découvre YouScribe en t'inscrivant gratuitement
Découvre YouScribe en t'inscrivant gratuitement
152
pages
Deutsch
Ebook
2005
Obtenez un accès à la bibliothèque pour le consulter en ligne En savoir plus
Publié par
Publié le
01 janvier 2005
Nombre de lectures
23
Langue
Deutsch
Poids de l'ouvrage
16 Mo
Publié par
Publié le
01 janvier 2005
Nombre de lectures
23
Langue
Deutsch
Poids de l'ouvrage
16 Mo
Dissertation zur Erlangung des Doktorgrades
der Fakultät für Chemie und Pharmazie
der Ludwig-Maximilians-Universität München
Molekulare Funktion und Regulation
des negativen Cofaktors 2, NC2
Elisa Piaia
aus
Montebelluna, Italien
2005Erklärung
Diese Dissertation wurde im Sinne von §13 Abs. 3 bzw. 4 der Promotionsordnung
vom 29. Januar 1998 von PD Dr. Michael Meisterernst betreut.
Ehrenwörtliche Versicherung
Diese Dissertation wurde selbstständig, ohne unerlaubte Hilfe erarbeitet.
München, 23.11.2004
Elisa Piaia
Dissertation eingereicht am: 23.11.2004
1. Gutachter: PD Dr. Michael Meisterernst
2. Gutachter: Prof. Dr. Patrick Cramer
Mündliche Prüfung am: 15.03.2005Molecular function and regulation
of the negative cofactor 2, NC2 a LinoTABLE OF CONTENTS
SUMMARY ..........................................................................................................1
I. INTRODUCTION ..............................................................................................2
1. Eukaryotic gene expression: from gene sequence to active protein ................................................... 2
2. Promoter structure in eukaryotic class II genes ................................................................................... 3
2.1. The core promoter elements .............................................................................................................. 3
2.1.1. TATA-box .................................................................................................................................. 3
2.1.2. Initiator Elements ...................................................................................................................... 4
2.1.3. DPE ........................................................................................................................................... 4
2.2. Regulatory sequences ........................................................................................................................ 4
2.2.1. Enhancer and Silencer elements ................................................................................................ 4
2.2.2. Insulator ..................................................................................................................................... 5
3. The preinitiation complex: the general transcription factors and the RNA Polymerase II ............. 5
3.1. The general transcription factors (GTFs) .......................................................................................... 5
3.1.1. TBP ............................................................................................................................................ 6
3.1.1.1. TBP paralogues .................................................................................................................. 6
3.1.2. TFIIB ......................................................................................................................................... 7
3.1.3. TFIIF 7
3.1.4. TFIIE 7
3.1.5. TFIIH 8
3.2. RNA Polymerase II ........................................................................................................................... 8
3.2.1. CTD ........................................................................................................................................... 9
3.2.2. The transcription cycle .............................................................................................................. 9
3.2.3. The holoenzyme ...................................................................................................................... 10
4. Transcriptional activators and repressors .......................................................................................... 11
5. Transcriptional cofactors/coregulators ............................................................................................... 11
5.1. TAFs: TBP associated factors ......................................................................................................... 12
5.1.1. TAF paralogues ....................................................................................................................... 12
5.2. TFIIA ............................................................................................................................................... 13
5.3. The Mediator ................................................................................................................................... 13
5.4. USA factors (upstream stimulatory activity) .................................................................................. 14
5.5. BTAF1 ............................................................................................................................................. 15
6. Chromatin structure ............................................................................................................................. 15
6.1. Histone modifications and histone modifying complexes .............................................................. 166.1.1. Acetylation and HATs/HDACs ................................................................................................ 17
6.1.2. Phosphorylation ....................................................................................................................... 17
6.1.3. Methylation and histone methyltransferases (HMTases) ........................................................ 17
6.1.4. Additional histones modifications ........................................................................................... 18
6.2. Non covalent chromatin modification: ATP-dependent Chromatin remodeling complexes ........... 18
7. The negative cofactor 2, NC2 ............................................................................................................... 19
7.1. NC2 is a general transcriptional repressor ...................................................................................... 22
7.2. Interplay between negative and positive effectors .......................................................................... 22
7.3. Positive role of NC2 in transcriptional regulation 23
8. Post-translational protein modifications: the role of phosphorylation in the regulation of
transcription factors ................................................................................................................................. 24
9. Silencing of transcription during mitosis ............................................................................................ 27
10. Protein transport to the nucleus ........................................................................................................ 31
10.1. The nuclear pore complex (NPC) ................................................................................................. 31
10.2. The nuclear localization signal (NLS) .......................................................................................... 32
II. RESULTS ......................................................................................................34
1. Localization studies of NC2.................................................................................................................. 34
1.1. Subcellular distribution of NC2 34
1.1.1. Localization of NC2 α ............................................................................................................. 34
1.1.2. The nucleolar localization of the endogenous NC2 α is restricted to the G1 phase ................ 36
1.1.3. Localization of NC2 β .............................................................................................................. 37
1.2. Import and dimerisation of NC2 ..................................................................................................... 37
1.2.1. Both NC2 α and NC2 β contain an NLS .................................................................................. 37
1.2.2. Is NC2 transported into the nucleus as a dimer or as single subunits? ................................... 40
1.3. Characterization of the NLS ........................................................................................................... 41
1.3.1. NC2 α has a bipartite NLS ....................................................................................................... 41
1.3.2. Mutation of threonine 23 in aspartate abolishes nulceolar localization .................................. 43
1.3.3. NC2 β has a single motif NLS ................................................................................................. 43
1.3.4. Only the first motif of the NC2 α-NLS is important for import of the dimer .......................... 45
1.3.5. Mutation of the NC2 β-NLS affect import of the dimer .......................................................... 45
2. Post-translational modification of NC2: NC2 α is specifically hyperphosphorylated during mitosis 47
2.1. NC2 is neither acetylated nor methylated ....................................................................................... 47
2.2. Monitoring of NC2 level throughout the cell cycle: a new isoform of NC2 α appears during mitosis
(M-NC2 α) .............................................................................................................................................. 49
2.3. The new M-NC2 α form is specific of the mitotic stage ..............................
