RNA polymerase I structure and transcription regulation:
Gespeichert in:
| 1. Verfasser: | |
|---|---|
| Format: | Abschlussarbeit Buch |
| Sprache: | English |
| Veröffentlicht: |
2013
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| Schlagworte: | |
| Online-Zugang: | Volltext Inhaltsverzeichnis |
| Beschreibung: | 104 Bl. Ill., graph. Darst. |
Internformat
MARC
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| 245 | 1 | 0 | |a RNA polymerase I structure and transcription regulation |c Christoph Engel |
| 264 | 1 | |c 2013 | |
| 300 | |a 104 Bl. |b Ill., graph. Darst. | ||
| 336 | |b txt |2 rdacontent | ||
| 337 | |b n |2 rdamedia | ||
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| 502 | |a München, Univ., Diss., 2014 | ||
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Datensatz im Suchindex
| _version_ | 1804152506415054848 |
|---|---|
| adam_text | TABLE OF CONTENTS
SUMMARY 3
ACKNOWLEDGMENTS 4
CONTRIBUTIONS 6
PUBLICATION 7
LIST OF FIGURES 8
TABLE OF CONTENTS 10
1. INTRODUCTION 13
1.1 GENERAL IMPORTANCE AND COMPOSITION OF RNA POLYMERASES 13
I
1.2 THE RDNA GENE AND ITS ORGANIZATION 14
1.3 POL I TRANSCRIPTION CYCLE 16
1.4 REGULATION OF POL I ACTIVITY 17
1.5 STRUCTURAL INFORMATION ON DNA-DEPENDENT RNA POLYMERASES AND THEIR
GENERAL
TRANSCRIPTION FACTORS 18
1.6 SCOPE OF THIS STUDY 21
2. MATERIALS AND METHODS 24
2.1 MATERIALS 24
2.1.1 STRAINS 24
2.1.2 OLIGONUCLEOTIDES 25
2.1.3 MEDIA 29
2.1.4 PLASMIDS 30
2.1.5 BUFFERS FOR THE PURIFICATION OF RNA POLYMERASE 1 33
2.2 MOLECULAR BIOLOGY METHODS 34
2.2.1 POLYMERASE CHAIN REACTION 34
2.2.2 PREPARATION OF COMPETENT E. COLI CELLS 34
2.2.3 TRANSFORMATION IN E. COLI AND PLASMID PURIFICATION 35
2.2.4 DNA DIGEST AND LIGATION 35
10
HTTP://D-NB.INFO/1059084716
2.2.5 PREPARATION OF COMPETENT YEAST CELLS AND DNA TRANSFORMATION 35
2.3 BIOCHEMICAL METHODS 37
2.3.1 FERMENTATION OF S. CEREVISIAE 37
2.3.2 FERMENTATION OF S. POMBE 37
2.3.3 POL I PURIFICATION 38
2.3.4 EXPRESSION AND PURIFICATION OF RECOMBINANT PROTEINS 39
2.3.5 PROTEIN ANALYSIS 39
2.3.6 TRANSCRIPTION ASSAYS 40
2.4 CRYSTALLIZATION AND STRUCTURE SOLUTION 40
2.4.1 POL I CRYSTALLIZATION AND MICROSEEDING 40
2.4.2 DATA COLLECTION AND PROCESSING 41
2.4.3 PHASING 41
2.4.4 MODEL BUILDING AND REFINEMENT 42
3. RESULTS AND DISCUSSION 43
3.1 PURIFICATION OF S. CEREVISIAE POL 1 43
3.2
(J
IS.
CEREVISIAE POL I CRYSTALLIZES IN THREE DIFFERENT FORMS, OF WHICH ONE IS
SUITABLE FOR
STRUCTURE DETERMINATION 43
*T
3.3 DATA COLLECTION AND PROCESSING 47
3.4 SOLVING THE STRUCTURE OF POL 1 48
3.5 POL I STRUCTURE 51
3.6 COMPOSITE ACTIVE CENTRE AND THE A49/A34.5 SUBCOMPLEX 54
3.7 EXPANDER AND CLEFT EXPANSION 55
3.8 CONNECTOR AND POLYMERASE DIMERIZATION 57
3.9 TRANSCRIPTION REGULATION 58
4. CONCLUSION AND OUTLOOK 61
4.1 CONCLUSIONS 61
4.2 EVALUATION OF PREVIOUSLY OBTAINED STRUCTURAL INFORMATION ON POL
1 62
4.3 RNA POLYMERASE I FROM AN EVOLUTIONARY POINT OF VIEW 64
11
4.4 FUTURE PERSPECTIVES 66
5. REFERENCES 70
6. APPENDIX 79
6.1 SCHIZOSACCHAROMYCES POMBE RNA POLYMERASE I 79
6.1.1 CONSTRUCTION AND CULTIVATION OF S. POMBE STRAINS WITH TAGGED POL I
SUBUNITS 79
6.1.2 S. POMBE POL I - PURIFICATION AND ACTIVITY 81
6.1.3 ATTEMPTS TO CRYSTALLIZE S. POMBE POL I 83
6.1.4 CONCLUSION OF THE S. POMBE POL I PROJECT 89
6.2 SCRIPTS FOR CRYSTALLOGRAPHIC DATA PROCESSING 91
6.2.1 XDS SCRIPT FOR DATA INTEGRATION 91
6.2.2 XSCALE SCRIPT FOR THE COMBINATION OF FIVE DATASETS FROM FOUR
CRYSTALS 92
6.2.3 XSCALE - SELECTED OUTPUT PARAMETERS 92
6.3 SCRIPTS AND PARAMETERIZATION FILES FOR STRUCTURE REFINEMENT 93
6.3.1 REFINEMENT STRATEGY FILE FOR PHENIX.REFINE 93
6.3.2 DEFINITION OF BOND LENGTHS FOR ONE COORDINATED ZINC ATOM 93
6.3.3 DEFINITION FILE FOR NCS REFINEMENT 94
6.3.4 DEFINITION FILE FOR RIGID BODY REFINEMENT 95
6.3.5 DEFINITION FILE FOR TLS REFINEMENT 96
6.3.6 SOLVENT DEFINITION FILE FOR REFINEMENT 96
6.4 STRUCTURE-BASED ALIGNMENT OF A190 AND RPBL 97
6.5 STRUCTURE-BASED ALIGNMENT OF A135 AND RPB2 98
6.6 STRUCTURE-BASED ALIGNMENT OF AC40/AC19 AND THE RPB3/RPBL 1
SUBCOMPLEX 99
6.7 CONSERVATION OF POL I AND POL II SUBUNITS 100
6.8 DOMAIN DIFFERENCES BETWEEN THE POLYMERASE SUBUNITS A190/RPBL AND
A135/RPB2 ..101
6.9 EVOLUTIONARY CONSERVATION OF EXPANDER AND CONNECTOR ELEMENTS 102
6.10 EXPANDER MUTATIONS DO NOT DISPLAY A GROWTH DEFECT IN S. CEREVISIAE
ON
YPD MEDIUM 103
7. ABBREVIATIONS 104
12
|
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| spelling | Engel, Christoph Verfasser aut RNA polymerase I structure and transcription regulation Christoph Engel 2013 104 Bl. Ill., graph. Darst. txt rdacontent n rdamedia nc rdacarrier München, Univ., Diss., 2014 (DE-588)4113937-9 Hochschulschrift gnd-content Erscheint auch als Online-Ausgabe urn:nbn:de:bvb:19-173904 https://nbn-resolving.org/urn:nbn:de:bvb:19-173904 Resolving-System kostenfrei Volltext DNB Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=027506262&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Engel, Christoph RNA polymerase I structure and transcription regulation |
| subject_GND | (DE-588)4113937-9 |
| title | RNA polymerase I structure and transcription regulation |
| title_auth | RNA polymerase I structure and transcription regulation |
| title_exact_search | RNA polymerase I structure and transcription regulation |
| title_full | RNA polymerase I structure and transcription regulation Christoph Engel |
| title_fullStr | RNA polymerase I structure and transcription regulation Christoph Engel |
| title_full_unstemmed | RNA polymerase I structure and transcription regulation Christoph Engel |
| title_short | RNA polymerase I structure and transcription regulation |
| title_sort | rna polymerase i structure and transcription regulation |
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