Biochemical studies of human TBP-associated factor TAF2 and a TAF2 and a TAF2-8-10 subcomplex of human general transcription factor TFIID Cristina Viola To cite this version: Cristina Viola. Biochemical studies of human TBP-associated factor TAF2 and a TAF2 and a TAF2- 8-10 subcomplex of human general transcription factor TFIID. Biochemistry, Molecular Biology. Uni- versité de Grenoble, 2014. English. NNT : 2014GRENV072. tel-01561609 HAL Id: tel-01561609 https://tel.archives-ouvertes.fr/tel-01561609 Submitted on 13 Jul 2017 HAL is a multi-disciplinary open access L’archive ouverte pluridisciplinaire HAL, est archive for the deposit and dissemination of sci- destinée au dépôt et à la diffusion de documents entific research documents, whether they are pub- scientifiques de niveau recherche, publiés ou non, lished or not. The documents may come from émanant des établissements d’enseignement et de teaching and research institutions in France or recherche français ou étrangers, des laboratoires abroad, or from public or private research centers. publics ou privés. THÈSE To obtain the title of / Pour obtenir le grade de DOCTEUR DE L’UNIVERSITÉ DE GRENOBLE Discipline / Spécialité : Doctorat CSV/Chimie Physique Moléculaire et Structurale Arrêté ministériel : Presented by / Présentée par : Cristina VIOLA Thesis supervisor / Thèse dirigée par : Prof. Imre BERGER Thesis prepared at / Thèse préparée au sein du European Molecular BIology Laboratory (EMBL) Grenoble Outstation In / dans : l'École Doctorale Chimie et Sciences du Vivantes Biochemical studies of human TBP-associated factor TAF2 and a TAF2-8-10 subcomplex of human general transcription factor TFIID. Thèse soutenue publiquement le 24 Octobre 2014 devant le jury composé de : Dr. Carlo PETOSA President/ Président Chef d'équipe, IBS Grenoble, France Prof. Andreas LADURNER Reviewer/ Rapporteur Professor, LMU Munchen, Allemagne Dr. Christophe ROMIER Reviewer/ Rapporteur Chef d'équipe, IGBMC Strasbourg, France Prof. Imre BERGER Directeur de Thèse Chef d'équipe, EMBL Grenoble, France Université Joseph Fourier / Université Pierre Mendès France / 1 Université Stendhal / Université de Savoie / Grenoble INP Table of Contents 1. Introduction ....................................................................................................................... 6 1.1. Eukaryotic Transcription, TFIID and the pre-initiation complex .................................. 10 1.2. Basal transcription, the core promoter and TFIID ........................................................... 11 1.2.1. Initiator element .............................................................................................................. 12 1.3. TSM-1 becomes TAF2(TFII150) a bona fide TAF conserved from Yeast to Humans .. 14 1.3.1. TAF2 core promoter INR recognition ............................................................................ 16 1.3.2. TAF2 in TFIID ................................................................................................................ 16 1.3.3. Domain Structure of TAF2 ............................................................................................. 17 1.4. Structural studies of TFIID ................................................................................................. 18 1.4.1. Structural analysis of TAFs ............................................................................................ 18 1.5. Production of TAF samples for functional and structural studies .................................. 18 1.5.1. Purification of native TAFs ............................................................................................ 18 1.5.2. Production of recombinant TAFs for structural and functional studies .......................... 19 1.6. Publications ........................................................................................................................... 21 1.6.1. Publication 1 ................................................................................................................... 21 1.6.2. Publication 2 ................................................................................................................... 37 1.6.3. Publication 3 ................................................................................................................... 49 2. Materials and methods ................................................................................................... 65 2.1. Materials ................................................................................................................................ 65 2.1.1. Chemicals ........................................................................................................................ 65 2.1.2. Enzymes and Antibodies ................................................................................................. 66 2.1.3. Plasmids .......................................................................................................................... 66 2.1.4. Oligonucleotides ............................................................................................................. 67 2.1.5. Cell Lines ........................................................................................................................ 67 2.1.6. Bacterial strains ............................................................................................................... 68 2 2.1.7. Crystallization Screens .................................................................................................... 68 2.1.8. Software and programs ................................................................................................... 68 2.2. Methods ................................................................................................................................. 69 2.3. Nucleic acid biochemistry .................................................................................................... 69 2.3.1. Concentration determination of nucleic acids ................................................................. 69 2.3.2. Preparation of plasmid DNA .......................................................................................... 69 2.3.3. Agarose gel electrophoresis ............................................................................................ 69 2.3.4. Restriction digestion of DNA ......................................................................................... 70 2.3.5. Polymerase chain reaction (PCR) ................................................................................... 70 2.3.6. DNA ligation ................................................................................................................... 70 2.3.7. DNA extraction from agarose gels ................................................................................. 71 2.3.8. DNA Sequencing ............................................................................................................ 71 2.4. General cloning strategies .................................................................................................... 71 2.4.1. Cloning TAF2 constructs ................................................................................................ 71 2.4.2. Cloning TAF8 constructs ................................................................................................ 74 2.4.3. Cloning TAF2-8-10 ........................................................................................................ 74 2.4.4. Overlap extension PCR ................................................................................................... 76 2.4.5. Sequence and ligation independent cloning .................................................................... 77 2.5. Protein production techniques ............................................................................................ 78 2.5.1. Baculovirus production and protein expression in insect cells ....................................... 78 2.5.2. Protein expression in E. coli ........................................................................................... 80 2.5.3. Purification of His-tagged protein from E. coli cytosolic fraction ................................. 80 2.5.4. Purification of TAF2 constructs and TAF2-8-10 complex from Sf21 lysate ................. 81 2.5.5. Expression of [ 13 C, 15 N]-labeled MBP-TAF8 C-terminal constructs ............................. 82 2.6. Protein Biochemistry and biochemical methods ............................................................... 82 2.6.1. Denaturing polyacrylamide gel electrophoresis (SDS-PAGE) ....................................... 82 2.6.2. Limited proteolysis ......................................................................................................... 83 2.6.3. Electrospray ionization mass spectrometry ..................................................................... 83 3 2.6.4. Edman sequencing .......................................................................................................... 84 2.6.5. Binding experiments using a peptide array ..................................................................... 84 2.7. Cells and cell culture ............................................................................................................ 85 2.7.1. Cultivation of E. coli cells .............................................................................................. 85 2.7.2. Transformation of chemically competent bacteria .........................................................