RNA polymerase I domain architecture and basis of rRNA cleavage:
Gespeichert in:
| 1. Verfasser: | |
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| Format: | Abschlussarbeit Buch |
| Sprache: | English |
| Veröffentlicht: |
2011
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| Schlagworte: | |
| Online-Zugang: | kostenfrei https://nbn-resolving.org/urn:nbn:de:bvb:19-146779 Inhaltsverzeichnis |
| Beschreibung: | 101, XXIII Bl. Ill., graph. Darst. |
Internformat
MARC
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| 245 | 1 | 0 | |a RNA polymerase I domain architecture and basis of rRNA cleavage |c Stefan Jennebach |
| 264 | 1 | |c 2011 | |
| 300 | |a 101, XXIII Bl. |b Ill., graph. Darst. | ||
| 336 | |b txt |2 rdacontent | ||
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| 502 | |a München, Univ., Diss., 2012 | ||
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Datensatz im Suchindex
| _version_ | 1804149399330226176 |
|---|---|
| adam_text | IMAGE 1
TABLE OF CONTENTS
V TABLE OF CONTENTS
I ACKNOWLEDGEMENT II
II SUMMARY IV
III CONTRIBUTIONS V
IV PUBLICATIONS VI
V TABLE O F CONTENTS VII
1 INTRODUCTION 1
1.1 TRANSCRIPTION SYSTEMS I N EUKARYOTES 1
1.1.1 EUKARYOTIC RNA POLYMERASES 1
1.1.2 SPECIFIC SUBUNITS IN POL I AND POL III 2
1.1.3 STRUCTURAL INFORMATION ON EUKARYOTIC POLYMERASES AND GTFS 4 1.2
TRANSCRIPTION O F RDNA AND RIBOSOME BIOGENESIS 5
1.2.1 RRNA IN RIBOSOMES 5
1.2.2 STRUCTURE OF THE NUCLEOLUS AND ORGANIZATION OF RIBOSOMAL DNA LOCI
6 1.2.3 EPIGENETICS OF RRNA GENES 7
1.2.4 THE POL I TRANSCRIPTION CYCLE 8
1.2.5 REGULATION OF RDNA TRANSCRIPTION IN VIVO 11
1.2.6 RIBOSOME ASSEMBLY 12
1.3 AIM O F THIS W O R K 14
2 MATERIALS AND METHODS 1 5
2.1 MATERIALS 15
2.1.1 BACTERIAL AND YEAST STRAINS 15
2.1.2 MEDIA 15
2.1.3 BUFFERS, MARKERS, SOLUTIONS AND ENZYMES 17
2.2 METHODS 2 1
2.2.1 PROTEIN EXPRESSION AND PURIFICATION 21
2.2.1.1 PURIFICATION OF POL I FROM S.CEREVISIAE 21
2.2.1.2 PREPARATION OF POL I AA49/A34.5 22
2.2.1.3 PREPARATION OF POL I A12.2AC 23
2.2.1.4 PREPARATION OF POL I IN COMPLEX WITH RRN3 23
2.2.1.5 PREPARATION OF RRN3 23
2.2.1.6 PREPARATION OF RECOMBINANT A49/A34.5 24
2.2.1.7 PURIFICATION OF POL III FROM S.CEREVISIAE 24
2.2.2 CRYSTALLIZATION OF POL I AND ATTEMPTS ON SOLVING THE STRUCTURE 26
2.2.2.1 CRYSTALLIZATION BY VAPOR DIFFUSION 26
2.2.2.2 STREAK-SEEDING 26
2.2.2.3 CRYSTAL HARVESTING AND CRYO-PROTECTION 27
2.2.2.4 HEAVY ATOM DERIVATIZATION AND CRYSTAL FREEZING 27
2.2.2.5 CRYSTALLIZATION OF POL I ELONGATION COMPLEXES 27
2.2.2.6 DATA COLLECTION 28
2.2.2.7 DATA PROCESSING 29
2.2.2.8 MOLECULAR REPLACEMENT 29
2.2.3 IN VITRO RNA ASSAYS 30
2.2.3.1 RNA EXTENSION ASSAYS USING NUCLEIC ACID SCAFFOLDS 30
2.2.3.2 RNA CLEAVAGE ASSAYS 31
2.2.4 CROSSLINKING AND MASS SPECTROMETRY 31
2.2.4.1 CHEMICAL CROSSLINKING OF MULTI-SUBUNIT PROTEIN COMPLEXES 3 1
2.2.4.2 SAMPLE PREPARATION FOR MS ANALYSIS 32
2.2.4.3 MASS SPECTROMETRY 32
VII
HTTP://D-NB.INFO/1025178831
IMAGE 2
TABLE OF CONTENTS
2.2.4.4 DATABASE SEARCHING 33
2.2.5 FIGURE PREPARATION 34
3 RESULTS AND DISCUSSION 3 5
3.1 PURIFICATION O F POL I VARIANTS 35
3.1.1 PURIFICATION OF POL I AA49/A34.5 35
3.1.2 PURIFICATION OF POL I A12.2AC 36
3.2 CRYSTALLIZATION O F POL I AND ATTEMPTS ON STRUCTURE SOLUTION 37
3.2.1 CRYSTALLIZATION OF COMPLETE POL 1 37
3.2.2 CRYSTALLIZATION OF POL I ELONGATION COMPLEXES 38
3.2.3 CRYSTALLIZATION OF POL I AA49/A34.5 40
3.2.4 CRYSTAL MANIPULATION AND HEAVY ATOM DERIVATIZATION 4 1
3.2.5 DATA COLLECTION AND PROCESSING 43
3.2.6 THE POL I ASYMMETRIC UNIT COMPRISES SEVEN POL I MOLECULES 44 3.2.7
ATTEMPTS ON STRUCTURE DETERMINATION BY MOLECULAR REPLACEMENT 45 3.3
CRYO-ELECTRON MICROSCOPIC STUDIES ON POL 1 4 7
3.3.1 CRYO-EM STRUCTURE OF POL I AT 12 A RESOLUTION 47
3.3.2 CRYO-EM STRUCTURE OF POL I AA49/A34.5 49
3.4 FUNCTIONAL STUDIES ON POL 1 50
3.4.1 A49/A34.5 ACTS AS A BUILD IN ELONGATION FACTOR 50
3.4.2 POL I HAS INTRINSIC CLEAVAGE ACTIVITY THAT REQUIRES A12.2 51
3.5 CROSSLINKING REVEALS POL I DOMAIN ARCHITECTURE AND POSITIONS THE POL
ISPECIFIC INITIATION FACTOR RRN3 54
3.5.1 CROSSLINKING-MS ANALYSIS OF POL 1 54
3.5.2 CONFIRMATION OF THE POL I CORE MODEL 54
3.5.3 MODEL EXTENSION REVEALS A UNIQUE JAW 56
3.5.4 A12.2 C-RIBBON BINDS PORE LIKE TFIIS 59
3.5.5 A49/A34.5 BINDS THE LOBE LIKE TFIIF 61
3.5.6 A49 TWH DOMAIN LIES ABOVE THE CLEFT 62
3.5.7 RRN3 BINDS POL I NEAR SUBCOMPLEX AC40/19 66
3.5.8 COMPARISON OF CROSSLINKING AND EM-DATA 67
3.6 CROSSLINKING ANALYSIS O F POL III 68
3.6.1 CROSSLINKING CONFIRMS THE POL III HOMOLOGY MODEL 68
3.6.2 EXTENSION OF THE POL III HOMOLOGY MODEL 70
3.6.3 C53/C37 BINDS THE LOBE LIKE TFIIF AND CONNECTS TO C11 72
3.6.4 LOCATION OF THE C82/34/31 SUBCOMPLEX 75
4 CONCLUSIONS AND OUTLOOK 7 9
5 REFERENCES 8 3
VI APPENDIX IX
VI.1 ALIGNMENTS O F SUBUNITS I N POL I AND T H E I R HOMOLOGS I N POL I
I IX
VI.2 ALIGNMENTS O F SUBUNITS I N POL III AND T H E I R HOMOLOGS I N POL
I I XIV
VI.3 CLONING AND EXPRESSION O F RRN7 XX
VII ABBREVIATIONS XXII
VIII CURRICULUM VITAE XXIII
VIII
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| dewey-ones | 572 - Biochemistry |
| dewey-raw | 572.8845 |
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| dewey-sort | 3572.8845 |
| dewey-tens | 570 - Biology |
| discipline | Biologie |
| format | Thesis Book |
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| spelling | Jennebach, Stefan 1981- Verfasser (DE-588)1024983897 aut RNA polymerase I domain architecture and basis of rRNA cleavage Stefan Jennebach 2011 101, XXIII Bl. Ill., graph. Darst. txt rdacontent n rdamedia nc rdacarrier München, Univ., Diss., 2012 (DE-588)4113937-9 Hochschulschrift gnd-content Erscheint auch als Online-Ausgabe urn:nbn:de:bvb:19-146779 http://edoc.ub.uni-muenchen.de/14677/ Verlag kostenfrei Volltext https://nbn-resolving.org/urn:nbn:de:bvb:19-146779 Resolving-System DNB Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=025212822&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Jennebach, Stefan 1981- RNA polymerase I domain architecture and basis of rRNA cleavage |
| subject_GND | (DE-588)4113937-9 |
| title | RNA polymerase I domain architecture and basis of rRNA cleavage |
| title_auth | RNA polymerase I domain architecture and basis of rRNA cleavage |
| title_exact_search | RNA polymerase I domain architecture and basis of rRNA cleavage |
| title_full | RNA polymerase I domain architecture and basis of rRNA cleavage Stefan Jennebach |
| title_fullStr | RNA polymerase I domain architecture and basis of rRNA cleavage Stefan Jennebach |
| title_full_unstemmed | RNA polymerase I domain architecture and basis of rRNA cleavage Stefan Jennebach |
| title_short | RNA polymerase I domain architecture and basis of rRNA cleavage |
| title_sort | rna polymerase i domain architecture and basis of rrna cleavage |
| topic_facet | Hochschulschrift |
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