„Functional analysis of multiple general transcription factors in Sulfolobus acidocaldarius” Dissertation Zur Erlangung des akademischen Grades eines Doktors der Naturwissenschaften - Dr. rer. nat. - vorgelegt von Bernadette Rauch geboren am 13.07.1984, in Dernbach Biofilm Centre, Molekulare Enzymtechnologie und Biochemie Fachbereich Chemie der Universität Duisburg-Essen 2013 Die vorliegende Arbeit wurde im Zeitraum von November 2009 bis Juli 2013 in der Arbeitsgruppe von Prof. Dr. Bettina Siebers in der Fakultät Chemie, Biofilm Centre, Molekulare Enzymtechnologie und Biochemie der Universität Duisburg-Essen durchgeführt. Tag der Disputation: 04.12.2013 Gutachter: Prof. Dr. Bettina Siebers Prof. Dr. Perihan Nalbant Vorsitzender: Prof. Dr. Ulbricht Table of contents Table of contents 1. Introduction .......................................................................................................................... 1 1.1. Archaea - an overview ................................................................................................. 1 1.2. Chromatin proteins in Eukaryotes and Archaea ........................................................... 2 1.3. Transcription apparatus in Bacteria, Eukaryotes and Archaea .................................... 2 1.4. RNAPs in the three domains of life ............................................................................. 5 1.5. Comparison of promoter structures in the three domains of life ................................. 7 1.6. Structure and function of transcription factors in Archaea and Eukaryotes ................ 8 1.6.1. The TATA-binding protein TBP .......................................................................... 8 1.6.2. Eukaryotic transcription factor TFIIB and its archaeal counterpart TFB ............ 8 1.6.3. Eukaryotic transcription factor TFIIE and its archaeal homolog TFE ................. 9 1.6.4. Multiple general transcription factors in Archaea and Eukaryotes .................... 10 1.7. Transcriptional regulation in Archaea ....................................................................... 11 1.8. Aims of this study ...................................................................................................... 12 2. Material and Methods ........................................................................................................ 13 2.1. Instruments ................................................................................................................. 13 2.2. Chemicals ................................................................................................................... 14 2.3. Plasmids ..................................................................................................................... 14 2.4. Strains and growth conditions .................................................................................... 14 2.5. Preparation of glycerol stocks of S. acidocaldarius .................................................. 16 2.6. Basic molecular biology methods .............................................................................. 16 2.6.1. Preparation of chromosomal DNA from S. acidocaldarius and quantification of DNA ............................................................................................................... 16 2.6.2. Isolation of plasmid DNA from E. coli .............................................................. 16 2.6.3. Agarose gel electrophoresis ............................................................................... 16 2.6.4. Amplification of DNA fragments via PCR ........................................................ 17 2.6.5. Restriction of DNA ............................................................................................ 17 2.6.6. Ligation of vector and insert .............................................................................. 17 2.6.7. Preparation of chemically competent E. coli cells ............................................. 17 2.6.8. Transformation of competent cells by heat-shock ............................................. 18 2.6.9. DNA sequencing ................................................................................................ 18 2.6.10. Sequence analysis ............................................................................................. 18 2.6.11. Gene synthesis .................................................................................................. 18 I Table of contents 2.7. Analysis of genome organization of tfb2 ................................................................... 18 2.7.1. Isolation of total RNA from S. acidocaldarius cells .......................................... 18 2.7.2. Removal of genomic DNA by DNaseI treatment .............................................. 19 2.7.3. First strand cDNA synthesis ............................................................................... 19 2.8. Basic biochemical methods ........................................................................................ 20 2.8.1. Protein quantification using Bradford reagent ................................................... 20 2.8.2. BC assay for protein quantification .................................................................... 20 2.8.3. SDS-polyacrylamide gel electrophoresis (SDS-PAGE) .................................... 20 2.9. Heterologous expression of transcription factors in E. coli ....................................... 21 2.9.1. Cloning of GTFs from S. acidocaldarius into overexpression vectors .............. 21 2.9.2. Expression and purification of SaTFB1 ............................................................. 24 2.9.3. Expression and purification of SaTFB2 and SaTFB3 ........................................ 24 2.9.4. Expression and purification of SaTBP ............................................................... 25 2.9.5. Expression and purification of SaTFE ............................................................... 25 2.9.6. Expression and purification of SaRpoG ............................................................. 26 2.10. Homologous expression using S. acidocaldarius MW001 as expression host ........ 26 2.10.1. Preparation of competent S. acidocaldarius MW001 cells .............................. 27 2.10.2. Electroporation of competent S. acidocaldarius MW001 cells ........................ 27 2.11. Purification of Strep-tag proteins ............................................................................. 28 2.12. Western blot and immunodetection ......................................................................... 28 2.12.1. Antibody generation and immunodetection ..................................................... 28 2.12.2. Antibody purification from antiserum using protein-A-agarose ...................... 28 2.12.3. Western blot and immunodetection (His-tag antibody) ................................... 29 2.12.4. Immunodetection using Strep-tag antibody ...................................................... 29 2.12.5. Immunodetection with FLAG-tag antibody ..................................................... 29 2.13. Construction of knock-out strains by homologous recombination and insertion of Sso-pyrEF cassette as genetic marker in S. acidocaldarius ............................... 30 2.13.1. Growth of the tfb3 disruption strain (tfb3::pyrEF) and S. acidocaldarius MW001 after UV-treatment ............................................................................. 33 2.14. Genomic knock-in of His-tag and Strep-FLAG tag ................................................. 33 2.14.1. Genomic tagging of transcription factors and subunit RpoG ........................... 33 2.14.2. Growing conditions of knock-in mutants and purification of transcription factors via genomic tags ................................................................................... 37 2.14.3. Growth of S. acidocaldarius MW001::rpoG-His and purification of the endogenous RNAP from S. acidocaldarius ...................................................... 37 II Table of contents 2.15. Reporter gene assays ................................................................................................ 37 2.15.1. Cloning of promoter regions of transcription factors for β-galactosidase reporter gene assays ......................................................................................... 37 2.15.2. Treatment of cultures for β-galactosidase reporter gene assays ....................... 38 2.15.3. Sample preparation for β-galactosidase reporter gene assays .......................... 38 2.16. Protein-protein interaction studies using the yeast two-hybrid system ................... 39 2.16.1. Construction of yeast two-hybrid vectors ......................................................... 39 2.16.2. Preparation of supercompetent E. coli DH5α cells .......................................... 41 2.16.3. Preparation of competent yeast cells ................................................................ 41 2.16.4. Transformation of yeast cells ........................................................................... 41 2.16.5. Protein-protein interaction studies .................................................................... 42 2.16.6. Testing false positive reactions ........................................................................ 42 2.16.7. Preparation of yeast cell extracts ...................................................................... 42 2.16.8. Urea/SDS-method ............................................................................................