Functional characterization of the COOH-terminal kinase activity of the TBP-associated factor TAF1 [Elektronische Ressource] / vorgelegt von Tobias Maile

Functional characterization of the COOH-terminal kinase
activity of the TBP-associated factor TAF1




Dissertation zur Erlangung des Doktorgrades
der Naturwissenschaften (Dr. rer. nat.)

Fakultät Naturwissenschaften
Universität Hohenheim

Institut für Genetik, Fg. Allgemeine Virologie
und
Department of Biochemistry,
University of California Riverside



vorgelegt von
Tobias Maile

aus Leonberg
2006





Dekan: Prof. Heinz Breer
1. berichtende Person: Prof. Frank Sauer
2. berichtende Person: Prof. Artur Pfitzner
Eingereicht am: 27. Februar 2006
Mündliche Püfung am: 13. Juli 2006





Die vorliegende Arbeit wurde am 3. Mai 2006 von der Fakultät Naturwissenschaften der
Universität Hohenheim als „Dissertation zur Erlangung des Doktorgrades der Natur-
wissenschaften“ angenommen.































Für meine Großeltern.

Erklärung

Hiermit versichere ich, daß ich die vorliegende Arbeit selbständig und mit den
angegebenen Quellen und Hilfsmittel angefertigt habe. Alle Ausführungen, die wörtlich
oder sinngemäß übernommen wurden, sind als solche gekennzeichnet.




Tobias Maile, Februar 2006








Dissertation der Fakultät Naturwissenschaften der Universität Hohenheim, erstellt extern
am Department of Biochemistry der University of California Riverside.



Gutachter:

Prof. Dr. Frank Sauer (University of California Riverside)

Prof. Dr. Artur Pfitzner (Universität Hohenheim) Contents I




I. Table of Contents

I. TABLE OF CONTENTS................................................................................................................................I
II. ABBREVIATIONS..................................................................................................................................... VI
1. INTRODUCTION...........1
1.1 THE COMPLEXITY OF TRANSCRIPTION REGULATION ......................................................................................1
1.1.1 Regulatory DNA sequences..................................................................................................................1
1.1.2 Promoter recognition and the basal transcription apparatus................................................................3
1.2 TFIID AND TAFS.........5
1.2.1 Function of TFIID in transcription.......................................................................................................5
1.2.2 DNA binding of TFIID.........................................................................................................................6
1.2.3 Different TFIID complexes ..................................................................................................................8
1.2.4 TAF1 is essential for the nucleating function of TFIID .........................................................................9
1.2.5 Activators regulate the function of TAF1............................................................................................11
1.2.6 TAF1 plays an essential role in promoter recognition ........................................................................12
1.2.7 TAF1 modifies GTFs and other proteins ............................................................................................13
1.3 CHROMATIN..............................................................................................................................................14
1.3.1 The structure of chromatin.................................................................................................................14
1.3.2 The dynamic nucleosome...................................................................................................................15
1.4 REGULATION OF CHROMATIN ACTIVITY......................................................................................................16
1.4.1 ATP-dependent chromatin-remodeling complexes..............................................................................17
1.4.2 Covalent modifications of histones: the “histone code”......................................................................17
1.4.2.1 Acetylation................................................................................................................................................. 19
1.4.2.2 Ubiquitination ............................................................................................................................................ 20
1.4.2.3 Methylation............................ 21
1.4.2.4 Phosphorylation ......................................................................................................................................... 21
1.4.2.5 Functions of histone modifications.............................................................................................................. 23
1.4.2.5.1 Histone modifications change the charge of histone tails ...................................................................... 23
1.4.2.5.2 Histone modifications can recruit chromatin binding proteins............................................................... 24
1.4.2.5.3 Crosstalk between different histone modifications................................................................................ 25
1.4.2.6 Histone code mechanisms: “binary switches” and “modification cassettes” .................................................. 26
1.4.2.7 TAF1 modifies histones.............................................................................................................................. 27
1.5 SPECIFIC AIMS OF THIS STUDY....................................................................................................................29 Contents II



2. MATERIALS AND METHODS..................................................................................................................30
2.1 MATERIALS ..............................................................................................................................................30
2.1.1 Laboratory Equipment.......................................................................................................................30
2.1.2 Consumables and Kits .......................................................................................................................31
2.1.3 Chemicals, Enzymes, Proteins and Molecular Weight Markers...........................................................32
2.1.4 Antibodies and Affinity Matrixes........................................................................................................33
2.1.5 Radioactive Nucleotides............33
2.1.6 Bacteria Stocks..................................................................................................................................34
2.1.7 Insect Cells........................................................................................................................................34
2.1.8 Oligonucleotides ...............................................................................................................................34
2.1.8.1 Oligonucleotides for inserting point-mutations with PCR............................................................................. 34
2.1.8.2 Oligonucleotides for cloning....................................................................................................................... 35
2.1.8.3 Oligonucleotides for RT-PCR-reactions ...................................................................................................... 35
2.1.8.4 Oligonucleotides for XChIP-reactions (all in 5’-3’ direction) ....................................................................... 35
2.1.8.5 Oligonucleotides for QPCR-reactions (5’-3’)............................................................................................... 36
2.1.8.6 Oligonucleotides for sequencing (5’-3’) ...................................................................................................... 36
2.1.8.7 Linker-Oligonucleotides............................................................................................................................. 37
2.1.9 Plasmids ...........................................................................................................................................38
2.1.9.1 Cloning- and expressionvectors:.................................................................................................................. 38
2.1.9.2 Constructed vectors for cloning and expression: .......................................................................................... 39
2.1.9.3 Cloned constructs....................................................................................................................................... 39
2.1.10 Baculoviruses for expression in Sf9-cellculture41
2.1.11 Proteins....................41
2.1.11.1 Histones and histone-peptides .....