Transcription Regulation in the hyperthermophilic crenarchaeon Thermoproteus tenax strain Kra1 [Elektronische Ressource] / vorgelegt von Jeannette Marrero Coto

Jeannette Marrero

Informations

• Publié par: universitat_duisburg-essen
• Publié le: 01 janvier 2010
• Nombre de lectures: 68
• Langue: English
• Poids de l'ouvrage: 2 Mo

Extrait

„Transcription Regulation in the hyperthermophilic
crenarchaeon Thermoproteus tenax strain Kra1”



Dissertation

zur Erlangung des akademischen Grades eines
Doktors der Naturwissenschaften
– Dr. rer. nat. –

vorgelegt von

Jeannette Marrero Coto

geboren am 27.09.1977, in Havana

Biofilm Centre, Molekulare Enzymtechnologie und Biochemie
Fachbereich Chemie
der
Universität Duisburg-Essen



2010 Die vorliegende Arbeit wurde im Zeitraum von November 2006 bis Oktober
2009 im Arbeitskreis von Prof. Dr. Bettina Siebers in der Fakultät Chemie,
Biofilm Centre, Molekulare Enzymtechnologie und Biochemie der Universität
Duisburg-Essen durchgeführt.

Tag der Disputation:



Gutachter: Prof. Dr. Bettina Siebers
Prof. Dr. Ann Ehrenhofer-Murray
Vorsitzender: Prof. Dr. G. Haberhauer



TABLE OF CONTEXT______________________________________________________

TABLE OF CONTENTS
1 1. INTRODUCTION...............................................................................
1.1 TRANSCRIPTION IN EUKARYOTES….……………………………... 2
1.2 TRATION IN ARCHAEA…...…... 3
1.3 MULTIPROTEIN BRIDGING FACTOR 1 (MBF1)………….…...…... 6
11 2. MATERIALS AND METHODS......................................................
2.1 CHEMICALS AND PLASMIDS……………………….………………... 11
2.2 INSTRUMENTS………..………………………………………............... 11
2.3 STRAINS OF Escherichia coli AND GROWTH CONDITIONS…........ 12
2.4. MOLECULAR BIOLOGICAL METHODS WITH DNA…………….. 13
2.4.1 Preparation of genomic DNA from T. tenax …………………………... 13
2.4.2 Isolation of plasmid DNA from E. coli……………….......................... 13
2.4.3 Quantification of DNA…………....................................................... 13
2.4.4 Agarose gel electrophoresis…………………….................................. 14
2.4.5 Purification of DNA fragments…………………………………………. 14
2.4.6 Amplification of genomic DNA and plasmid DNA by PCR…………… 14
2.4.7 Enzymatic manipulation of DNA…………......................................... 16
2.4.7.1 Restriction of DNA…………..………………………..................... 16
2.4.7.2 Ligation of vector and insert………………………..………………. 16
2.4.8 Transformation…………………………………………….................... 16
2.4.8.1 Preparation of chemically competent E. coli cells…….................... 16
2.4.8.2 Transformation of the competent E. coli cells……......................... 17
2.4.8.3 Preparation of electrocnt E. coli cells…………..................... 17
2.4.8.4 Electroporation of electrocompetent E. coli cells…………………... 17
2.4.9 Molecular cloning of TFBs from T. tenax ……………………………... 17
2.4.10 Molecular cloning of wild-type T. tenax promoter regions and
mutated fba-pfp promoter fragments.……………………….……………………. 18
2.4.11 Automated DNA sequencing …………………………………………. 19
2.4.12 Labeling of DNA probes………………………………………………. 19
32 2.4.12.1 Oligonucleotide end-labeling with p-γ-ATP…………………….. 19
32 2.4.12.2 P-γ-ATP 5´ end-labeling of double stranded DNA probes……… 19
2.4.13 Oligonucleotide hybridisation to construct 60 bp-length dsDNA…….. 19
2.5 BIOCHEMICAL METHODS.............................................................. 20
2.5.1 Heterologous expression of the recombinant proteins in E. coli……….. 20
2.5.1.1 Expression and purification of Ttx-TFB1…………….…………….. 20
2.5.1.2 Expression aification of Ttx-TFB2……………………. 21
2.5.1.3 Expression and purification of Ttx-TBP...………………………….. 21
2.5.1.4 Expression aification of 6xHis-Ttx-TFB2…….……………... 22
2.5.1.5 Expression and purification of 6xHis-Ttx-TFB3…………………… 22
2.5.1.6 Expression aification of 6xHis-Ttx-MBF1…….. 23
2.5.2 Analytical protein methods…………………………………………….. 23
2.5.2.1 Protein quantification…………………………………................... 23
2.5.2.2 SDS-polyacrylamide gel electrophoresis (SDS-PAGE)..................... 23
2.5.2.3 Generation of antibodies using purified protein………..................... 24
2.5.2.4 Western blotting analysis........................……………….................... 25
2.6 RADIOACTIVE ELECTROPHORESIS MOBILITY SHIFT ASSAY.. 25
2.7 EXONUCLEASE III FOOTPRINTING ASSAY……………………….. 26
27 2.8 WORKING WITH YEAST AS GENETIC MODEL…………………...
2.8.1 Yeast strains……………………….……………………………………. 27 TABLE OF CONTEXT______________________________________________________

2.8.2 Preparation of competent yeast cells.…………..……………………….. 27
2.8.3 Transformation of yeast competent cells.………………..……………... 27
2.8.4 Preparation of genomic DNA from yeast using Winston solution……... 28
2.8.5 Recombination/gap repair cloning technique for cloning of the mbf1
gene from T. tenax, M. mazei and for the construction of chimeric
genes…………………………………………………………………... 28
2.8.6 Complementation test: sensitivity to aminotriazole……….................... 31
2.9 SEQUENCE ANALYSIS…………………………………..……………... 31
33 3. RESULTS……………………………………………………………………
3.1 SEQUENCE AND GENE CONTEXT ANALYSIS OF TFBs
IN T. tenax………………………...…………………….........................………... 33
3.2 HETEROLOGOUS EXPRESSION OF T. tenax GTFs IN E. coli........... 46
47 3.3 PROTEIN ENRICHMENT AND PURIFICATION……………......…..
3.4 DNA BINDING OF T. tenax GTFs………….....…………...................... 50
3.4.1 DNA binding of Ttx-TFB1 and Ttx-TFB2 using a large-length fba-pfp
promoter fragment……………………........……………………..…… 50
3.4.2 DNA binding of Ttx-TFB1, Ttx-TFB2 and Ttx-TFB3 using a short-
53 length fba-pfp promoter fragment....................................................
3.4.3 Influence of competitor DNA on the DNA Binding of Ttx-TFB2
to the fba-pfpWT-60bp……………………………………………………………. 55
3.4.4 DNA binding of Ttx-TFB1 and Ttx-TFB2 to mutated fba-pfp promoter
fragments....... ………………………………………………………… 55
3.4.5 Identification of Ttx-TFB1/Ttx-TBP and Ttx-TFB2 binding sites at the
fba-pfp promoter region………………………..................................... 57
3.5 EUKARYOTIC AND ARCHAEAL MBF1: A COMPARISON………. 62
3.5.1 Heterologous expression and purification of the T. tenax MBF1……… 70
3.5.2 Complementation of yeast MBF1 by archaeal MBF1.…….…………... 71
3.5.3 Chimera yeast - Archaeal MBF1 variants……………………………… 72
76 4. DISCUSSION………………………………………………………………
4.1 BIOINFORMATIC ANALYSIS OF CRENARHAEAL TFBs………... 76
4.2 CHARACTERIZATION OF TFBs FROM T. tenax……………….….. 81
4.3 MULTIPROTEIN BRIDGING FACTOR 1………………………...….. 87
92 5. SUMMARY…………………………………………….............................
94 6. LITERATURE………………………………………..……………………
100 7. APPENDIX………............................
100 7.1 LIST OF ABBREVIATIONS…………….………………………………
7.2 LIST OF PUBLICATIONS………………..…………………………….. 102
7.3 LEBENSLAUF………..……………………………...…………………… 104
7.4 ERKLÄRUNG ……………………………………………………………. 105
7.5 ACKNOWLEDGEMENTS………….....………………………………... 106 TABLE OF CONTEXT______________________________________________________

LIST OF FIGURES

29 FIGURE 1. Basics of gap-repair cloning in yeast with one insert and two inserts…………..
35 FIGURE 2. Phylogenetic tree for 46 TFB homologues in Crenarchaeota…………………..
37 FIGURE 3. Multiple sequence alignment of TFB1-4 homologues in Crenarchaeota……….
FIGURE 4. Domain and structural analysis of TFB1 and TFB2 homologues in
44 Crenarchaeota…………………………………………………………………………
45 FIGURE 5. Genome context analysis of TFB1- 4 in archaeal genomes…………………….
FIGURE 6. SDS-PAGE and western blot analysis of the purified recombinant 6xHis-Ttx-
48 TFB2…………………………………………………………………………….
49 FIGURE 7. SDS-PAGE of the purified recombinant 6xHis-Ttx-TFB3.……
50 FIGURE 8. SDS-PAGE and western blot analysis of the purified recombinant Ttx-TBP..…
51 FIGURE 9. DNA binding of Ttx-TFB1 in the presence and absence of Ttx-TBP…………..
FIGURE 10. DNA sequence of the fba-pfp, pps, orf1155-lrp, gar1-tfb2, and tfb1 probes
52 used for analysis of Ttx-TFB1 and Ttx-TFB2 binding………………………………..
53 FIGURE 11. DNA binding of Ttx-TFB2 in the presence and absence of Ttx-TBP………….
FIGURE 12. DNA binding of Ttx-TFB1, Ttx-TFB2 and Ttx-TFB3 to the fba-pfpWT-60bp
54 probe in the presence and absence of Ttx-TBP……………………………………….
FIGURE 13. DNA binding of Ttx-TFB2 to the fba-pfpWT-60bp probe in the presence and
55 absence of competitor DNA…………………………………………………………..
56 FIGURE 14. DNA sequence of fba-pfpWT and the mutated fba-pfp promoter regions……..
FIGURE 15. DNA binding of Ttx-TFB1 and Ttx-TFB2 to mutated promoter regions of the
57 fba-pfp operon………………………………………………………………………...
59 FIGURE 16. Principle of Exonuclease III footprinting technique…………………………..
FIGURE 17. Identification of Ttx-TFB1 and Ttx-TFB2 binding sites at the promoter region
60 of the fba-pfp operon from T. tenax…………………………………………………..
FIGURE 18. Summary of Ttx-TFB1/Ttx-TBP and Ttx-TFB2 -binding sites in the promoter
61 region of fba-pfp operon from T. tenax determined by Exo III footprinting assays….
FIG