Structure of the Human Transcription Factor TFIIF

Research Collection Doctoral Thesis Structure of the human transcription factor TFIIF Author(s): Gaiser, Florian Publication Date: 2000 Permanent Link: https://doi.org/10.3929/ethz-a-003928353 Rights / License: In Copyright - Non-Commercial Use Permitted This page was generated automatically upon download from the ETH Zurich Research Collection. For more information please consult the Terms of use. ETH Library Diss. ETI! No. 13735 Structure of the human ranscription factor TFIIF A dissertaiion submitted to the Swiss Federal Institute of Technolog} Zurich for the degree of Doctoi ol Natural Sciences presented by Florian Gaiscr Dipl. Narw.'ETH born May 29, 1972 from Munich (Germany) Prof. Dr. T. J. Richmond, cxatmnei Prof. Dr. M. Gruttei. coexammet Zurich, 2000 il Table of contents Table of contents ,...ii List of figures ....vi List of tables viii Abstract x Zusammenfassung , xi Abbreviations xii 1 introduction 1 1.1 Eukaryotic gene expression and the role of transcription 1 1.2 The Role of TFIIF in RNA polymerase II transcription 4 1.2.1 Assembly of the RNA polymerase II preinitiation complex (PIC) 4 1.2.2 The RNA polymerase II holoenzyme 12 1.2.3 Initiation of phosphodiester bond formation by RNA polymerase II 13 1.2.4 Transcript elongation by RNA polymerase II 20 1.2.5 TFIIF in activated transcription 24 1.3 Domain structure of human TFIIF 28 1.3.1 Domain structure of RAP74 28 1.3.2 Domain structure of RAP30 29 1.4 Outline of the thesis project and its goals 31 2 Materials and Methods. 33 2.1 Materials and Apparatus 33 2.1.1 Sources of general chemicals 33 2.1.2 Purification of polyethylene glycol (PEG) 36 2.1.3 Buffers and solutions 37 2.1.4 Media for bacterial growth 39 2.1.5 Enzymes for DNA-subcloning 39 2.1.6 Proteolytic enzymes 39 2.1.7 General apparatus 40 2.1.8 HPLC equipment.. 41 2.1.9 FPLC equipment 41 2.1.10 Centrifuges 41 2.1.11 Heavy atom compounds 42 2.1.12 Crystallization, X-ray sources and X-ray detection 42 2.1.13 Computing ...43 2.2 DNA analysis 45 2.2.1 Concentration 45 ni 2.2.2 DNA Polyacrylamide gel electrophoresis (DNA PAGE) 45 2.2.3 Electrophoretic DNA markers 45 2.3 DNA purification 46 2.3.1 Ethanol precipitation of DNA fragments 46 2.3.2 Spun column purification 46 2.3.3 Purification of synthetic oligonucleotides by denaturing gel electrophoresis 46 2.3.4 Agarose gel purification 47 2.3.5 Medium scale alkaline lysis plasmid preparation 47 2.4 Cloning methods 48 2.4.1 Competent cell preparation 48 2.4.2 Restriction digestion 48 2 4.3 Dephosphorylation 49 2.4.4 Ligation 49 2 4.5 Plasmid transformation 49 2.4.6 Design of PCR primers 49 2 4.7 PCR subcloning with cohesive-ended inserts 50 2.4.8 PCR subcloning with blunt-end inserts 50 2.4.9 PCR-subcloning based site directed mutagenesis (PCR-SDM) 51 2.4 10 DNA sequencing .....54 2.4.11 PCR screening .55 2.4.12 Small scale alkaline lysis (miniprep) 56 2.5 Protein analysis 57 2.5.1 Concentration ........57 2.5.2 SDS Polyacrylamide gel electrophoresis (SDS PAGE) 57 2.5.3 Electroblotting and N-terminal sequencing 57 2.5.4 Mass spectrometry ...58 2.5.5 Limited proteolysis 59 2.5.6 Analytical gel filtration 58 2.5.7 Dynamic light scattering 62 2.5.8 Ellmann's assay.... 62 2.6 Protein expression methods 63 2.6.1 Small scale expression test in 2xTY-media 63 2.6.2 Large (6 I) scale expression in 2xTY-media 63 2.6.3 Small scale expression test in SeMet-M9-media 65 2.6.4 Large (3 I) scale expression in SeMet-M9-media 65 2.6.5 Solubility test 66 2.7 Protein purification methods ...67 IV 2.7.1 Purification of human RAP74(2-517) 67 2.7.2 Purification of the C-terminal domain of human RAP74(364-517) 69 2.7.3 Purification of the N-terminal domain of human RAP74 71 2.7.4 Purification of human RAP30 and its N-terminal domain 74 2.7.5 Refolding and purification of human TFIIF 76 2.7.6 Refolding and purification of the RAP30/74-interaction domains 77 2.8 Crystallization screens .79 2.9 MeHgN03-prelabelling of TFIIF .83 3 Design of crystallization constructs based on human TFIIF domain structure by limited proteolysis 84 3.1 General scheme for limited proteolysic analysis 84 3.2 Results of limited endoproteolysis 87 3.3 Results of limited Exoproteolysis 93 3.4 Design of crystallization constructs based on the approximate domain structure of human TFIIF by endoproteolysis 97 3.5 Design of more cystallization constructs based on precise definition of domain boudaries by exoproteolysis 102 4 Crystallization of the RAP30/74-interaction domains of human TFIIF 105 4.1 Introduction of a general scheme for crystallization screening 105 4.2 Preparation of native crystals and collection of native data 112 4.2.1 Initial crystallization screening 112 4.2.2 Refinement of initial crystallization conditions 115 4.2.3 Cryoprotection screening and flash cooling 120 4.2.4 Dehydration screening.... 122 4.2.5 Collection of native data for RAP30(2-119)/RAP74(2-158) 125 4.2.6 Collection of native data for RAP30(2-119)/RAP74(2-154) 126 4.2.7 Collection of native data for RAP30(2-119)/RAP74(2~172) ....127 4.3 Discussion of crystallization screening and data collection 129 5 Screening for heavy atom derivatives for the RAP30/74-interaction domains of human TFIIF 135 5.1 Introduction of a general scheme for derivative screening 135 5.2 Derivative crystal preparation and derivative data collection 138 5.2.1 Derivative crystal screening by heavy atom compound soaking 138 5.2.2 Derivative crystal screening by heavy atom compound cocrystallization 141 5.2.3 Investigation of cysteine-alanine mutant proteins for derivative screening 141 V 5.2.4 Selenomethionine in vivo prelabeling of wild type and methionine- mutant proteins 142 5.2.5 Methylmercury in vitro prelabeling 146 5.3 Discussion of derivative screening and data collection..... 150 6 Crystal structure of the RAP30/74 interaction domains of human TFIIF 153 6.1 Overview of the structure determination process from phase determination to model validation , 153 6.2 Phase determination and phase improvement by density modification and phase cycling 155 6.3 Model building and refinement 162 6.4 A novel heterodimerization fold of the RAP30/74 interaction domains of human TFIIF at 1.7 À resolution 168 6.4.1 An novel "triple barrel" heterodimerization fold 168 6.4.2 The RAP74 "arm domain" 175 6.4.3 The C-terminal a-helix of RAP74(2-172) 176 7 Conclusions and future perspectives 178 References 182 Appendix 195 Human RAP74 amino acid and DNA sequences 195 Human RAP30 amino acid and DNA sequences 197 Multiple sequence alignment of RAP74 holomogues..... 199 Multiple sequence alignment of RAP30 holomogues 199 Acknowledgements 200 Curriculum Vitae 201 VI List of figures Figure 1: Basal transcription by RNA polymerase 11 3 Figure 2: Structures of the RNA polymerase 11 transcription machinery 19 Figure 3: Domain structure of human TFIIF 30 4: PCR based site-directed Figure mutagenesis , 52 Figure 5: Limited proteolysis assays 59 Figure 6: General scheme for limited proteolytic analysis 84 Figure 7: Initial RUNNlNG-assays with various endoproteases 87 Figure 8: Inhibitor testing with STOPPING-assay 88 Figure 9:Efficiency of inhibition and relative stability of TFT1F proteolytic fragments. ..89 Figure 10: HPLC-MS-analysis of chymotryptic digestion product 91 Figure 11: Initial RUNNING- and STOPPING-assays with carboxypeptidases 94 Figure 12: Comparison of various reaction conditions for exproteascs 95 Figure 13: Design of N-terminal RAP30 and RAP74 constructs for crystallization 101 Figure 14: General scheme of crystallization screening 106 Figure 15: Refinement of crystallization conditions: RAP30(2-119VRAP74(2-172) 116 Figure 16: Crystals of RAP30(2-119VRAP74(2-154). RAP30(2-119)/RAP74(2-158) and alignment of the unit cell axes b and b" with crystal morphology 117 Figure 17: Crystal forms of RAP30/74 interaction domain of human TFIIF before and after dehydration 133 Figure 18: Radiation damage during high resolution data collection of native of RAP30(2-119)/RAP74(2-172) 134 Figure 19: General scheme of derivative screening 135 Figure 20: Methylmercury prelabcled crystals of RAP30(2-119)/RAP74(2-154) 147 21 Figure : XFAS-expenmcnt and theoretical plot the mercury anomalous scattering components 148 Figure 22: Stability of crystalline diffraction limits during MAD-data collection 152 Figure 23: Process of structure determination for RAP30/74 interaction domains of human TFIIF 154 Figure 24. Anomalous difference patterson map 156 Figure 25: Vector recombination diagram 157 Figure 26: Phasing Power and R of final heavy atom model 159 Vil Figure 27: Fiïcct of heavy atom parameter refinement in MLPHARE and density modification with DM. 161 Figure 28: B-faclor distribution of the water molecules 164 Figure 29: Mean Ca-B-factors and mean NCS-C a-distanccs for the RAP30/74- intcraction domains of human TFIIF after superposition 166 Figure 30: Structure of RAP30/74-interaction domains of human TFIIF 169 Figure 3 I: Electron density maps from the '"triple barrel" core 170 Figure 32: Superpostition of the four copies of the RAP30/74-interactiondomam 171 Figure 33: Solvent accessible surfaces 173 Figure 34: Sequence alignments of RAP30C-119) and RAP74Q-172) 174 Figure 35: Functional aspects of the RAP50/74-interaction domains 176 36: Molecular of Figure weight RAP30/74-interaction domain complexes as determined by gel filtration and dvnamie light scattering 180 Mil List of tables Table 1 The general ttanscnption factors ol human RNA polymerase II 11 Table 2 Cloning sttams and plasmids 48 Table ^ Sequenung-Ptimeis ->4 1 able 4 PCR-Pumeis -yS Tablet 1 ndopioteases 61 Table

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