Electron microscopic location of tagged proteins in the yeast S. cerevisiae spliceosomal B and B act complexes:
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| Format: | Abschlussarbeit Buch |
| Sprache: | English |
| Veröffentlicht: |
2014
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| Schlagworte: | |
| Online-Zugang: | Inhaltsverzeichnis |
| Beschreibung: | IV, 123 S. Ill., graph. Darst. |
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| adam_text | AUS DEM MAX-PLANCK-INSTITUT FIIR BIOPHYSIKALISCHE CHEMIE IN GOTTINGEN
ABTEILUNG ZELLULARE BIOCHEMIE
DIREKTOR: PROF. DR.
REINHARD LIIHRMANN
ELECTRON-MICROSCOPIC LOCATION OF TAGGED PROTEINS IN
THE YEAST S. CEREVISIAE SPLICEOSOMAL B AND B
ACT
COMPLEXES
DISSERTATION
ZUR ERLANGUNG DES DOKTORGRADES
DER FAKULTAT FUR BIOLOGIE, CHEMIE UND GEOWISSENSCHAFTEN
DER UNIVERSITAT BAYREUTH
VORGELEGT VON
NORBERT RIGO
AUS AUGSBURG
BAYREUTH 2014
HTTP://D-NB.INFO/1073220834
TABLE OF CONTENTS I
TABLE OF CONTENTS
ABSTRACT 1
ZUSAMMENFASSUNG 4
1 INTRODUCTION 7
1.1 EUKARYOTIC GENE STRUCTURE AND PRE-MRNA SPLICING 7
1.2 PRE-MRNA SPLICING 8
1.2.1 THE CHEMISTRY OF THE SPLICING REACTION 8
1.2.2 SMALL NUCLEAR RIBONUCLEOPROTEINS (SNRNPS) - THE BUILDING BLOCKS OF
THE
SPLICEOSOME 9
1.2.3 SPLICEOSOME ASSEMBLY AND THE SPLICING CYCLE 13
1.2.4 PROTEOME OF THE SPLICEOSOME 15
1.2.5 REARRANGEMENT OF THE RNA NETWORK DURING SPLICING 19
1.3 STRUCTURAL ANALYSIS OF THE SPLICEOSOME USING ELECTRON MICROSCOPY 21
1.3.1 LOCATION OF RNA AND PROTEINS WITHIN THE SPLICEOSOME 23
1.4 AIMS OF THIS STUDY 23
2 MATERIALS AND METHODS 25
2.1 MATERIALS 25
2.1.1 CHEMICALS AND MEDIA 25
2.1.2 NUCLEOTIDES 26
2.1.3 PRE-MRNA CONSTRUCTS 27
2.1.4 DNA OLIGONUCLEOTIDES 28
2.1.5 ENZYMES AND ENZYME INHIBITORS 28
2.1.6 ANTIBODIES 29
2.1.7 PLASMIDS 29
2.1.8 ESCHERICHIA COLI STRAINS 30
2.1.9 SACCHAROMYCES CEREVISIAE STRAINS 30
2.1.10 HUMAN CELL LINES 30
2.1.11 COMMON BUFFERS AND MEDIA 31
2.1.12 KITS 33
2.1.13 MATERIALS FOR CHROMATOGRAPHY AND CENTRIFUGATION 34
2.1.14 MATERIALS FOR ELECTRON MICROSCOPY 34
2.1.15 DEVICES 34
2.2 METHODS 36
2.2.1 DETERMINATION OF DNA CONCENTRATIONS 36
II
TART .F. OF CONTENTS
2.2.2 DETERMINATION OF PROTEIN CONCENTRATION IN SOLUTION 36
2.2.3 DENATURING POLYACRYLAMIDE GEL ELECTROPHORESIS (PAGE) OF RNA 36
2.2.4 PROTEIN ANALYSIS VIA DENATURING PAGE 37
2.2.5 PRODUCTION OF ELECTRO-COMPETENT CELLS FOR ELECTROPORATION 37
2.2.6 ELECTROPORATION 38
2.2.7 PLASMID PREPARATION 38
2.2.8 PHENOL / CHLOROFORM / ISOAMYL ALCOHOL EXTRACTION AND ETHANOL
PRECIPITATION OF
NUCLEIC ACIDS 38
2.2.9 RESTRICTION DIGESTION OF PLASMIDS 39
2.2.10 IN VITRO TRANSCRIPTION 39
2.2.11 HYDROLYSIS OF PROTEINS WITHIN RNP COMPLEXES USING PROTEINASE K 41
2.2.12 OVEREXPRESSION AND PURIFICATION OF THE MS2-MBP FUSION PROTEIN 41
2.2.12.1 OVEREXPRESSION 41
2.2.12.2 PURIFICATION OF THE MS2-MBP FUSION PROTEIN 42
2.2.13 PREPARATION OF SPLICING-ACTIVE HELA NUCLEAR EXTRACT 42
2.2.14 SPLICING ACTIVITY TEST OF HELA NUCLEAR EXTRACT 43
2.2.15 PREPARATION OF SPLICING ACTIVE YEAST EXTRACT 44
2.2.16 SPLICING ACTIVITY TEST OF YEAST EXTRACT 45
2.2.17 IMMUNOBLOT 45
2.2.18 IN VITRO ASSEMBLY AND PURIFICATION OF HUMAN SPLICEOSOMAL C
COMPLEX 46
2.2.18.1 IN VITRO SPLICING REACTION 46
2.2.18.2 DENSITY-GRADIENT ULTRACENTRIFUGATION 46
2.2.18.3 AFFINITY CHROMATOGRAPHY ON AMYLOSE-AGAROSE 47
2.2.19 PURIFICATION OF YEAST SPLICEOSOMAL B COMPLEX 47
2.2.19.1 IN VITRO ASSEMBLY OF YEAST SPLICEOSOMAL B COMPLEXES 47
2.2.19.2 PURIFICATION OF YEAST B COMPLEX 48
2.2.20 PURIFICATION OF YEAST SPLICEOSOMAL B
ACT
COMPLEX 48
2.2.21 IMMUNOPRECIPITATION OF SPLICEOSOMAL COMPLEXES 49
2.2.22 IMMUNO-LABELING OF SPLICEOSOMAL COMPLEXES 49
2.2.22.1 IMMUNO-LABELING OF HUMAN C COMPLEXES 50
2.2.22.2 IMMUNO-LABELING OF YEAST SPLICEOSOMAL COMPLEXES 50
2.2.23 GRAFIX 50
2.2.23.1 GRAFIX WITH HUMAN C COMPLEXES 51
2.2.23.2 GRAFIX USED FOR CROSS-LINKING YEAST SPLICEOSOMES 51
2.2.24 ENZYME-LINKED IMMUNOSORBENT ASSAY 51
2.2.25 PREPARATION OF GRIDS FOR USE IN ELECTRON MICROSCOPY 52
2.2.26 PREPARATION OF THIN CARBON FILMS FOR SAMPLE ADSORPTION 53
2.2.27 SAMPLE PREPARATION FOR NEGATIVE-STAIN EM 53
TABLE OF CONTENTS U1
3 RESULTS 54
3.1 LOCATION ATTEMPTS IN HUMAN SPLICEOSOMAL COMPLEXES 54
3.1.1 PURIFICATION OF HUMAN FUNCTIONALLY ACTIVE STEP I SPLICEOSOMES 54
3.1.2 IMMUNO-LOCATION STRATEGY USING THE FLAG/HA-TAG 58
3.1.3 OTHER TAG-SYSTEMS FOR IMMUNO-LOCATION ATTEMPTS 64
3.1.3.1 FLAG/STREPII-TAG AND EGFP 64
3.2 LOCATION OF PROTEINS IN PURIFIED SPLICEOSOMES FROM S. CEREVISIAE 67
3.2.1 UTILIZATION OF THE TANDEM AFFINITY-PURIFICATION TAG FOR LOCATING
PROTEINS 68
3.2.2 YEAST PRE-CATALYTIC SPLICEOSOME 69
3.2.2.1 PURIFICATION OF YEAST PRE-CATALYTIC SPLICEOSOMES 71
3.2.2.2 LOCATION OF THE U4/U6 DI-SNRNP PROTEIN PRP3 73
3.2.2.3 LOCATION OF THE C-TERMINUS OF THE U5 SNRNP PROTEIN BRR2 76
3.2.2.4 LOCATION OF THE C-TERMINI OF THE U5 SNRNP PROTEINS PRP8 AND
SNULL4 77
3.2.2.5 SUMMARY OF THE LOCATION OF PRP8, BRR2, SNUL 14 AND PRP3 WITHIN
THE YEAST
B COMPLEX 80
3.2.3 YEAST ACTIVATED SPLICEOSOME 80
3.2.3.1 PURIFICATION OF YEAST ACTIVATED SPLICEOSOMES 81
3.2.3.2 LOCATION OF THE NTC-RELATED PROTEIN CWC2 82
3.2.3.3 LOCATION OF THE SPLICING FACTOR PRP2 85
3.2.3.4 LOCATION OF THE C-TERMINI OF THE U5 SNRNP PROTEINS BRR2, PRP8
AND SNUL 14 87
3.2.3.5 SUMMARY OF THE LOCATION OF PROTEINS WITHIN THE YEAST B
ACT
COMPLEX 90
4 DISCUSSION 92
4.1 ATTEMPTS TO LOCATE PROTEINS WITHIN THE HUMAN SPLICEOSOMAL C COMPLEX
92
4.2 LOCATION OF PROTEINS WITHIN THE YEAST B COMPLEX 94
4.2.1 FITTING OF THE YEAST TRI-SNRNP INTO THE YEAST B COMPLEX 96
4.2.2 THE COMPARISON OF THE HUMAN WITH THE YEAST B COMPLEX REVEALS THE
POSITION OF
THE PRE-MRNA AND THE U1 AND U2 SNRNP 98
4.2.3 FUNCTIONAL IMPLICATIONS OF THE LOCATION OF THE U5-SPECIFIC
PROTEINS 99
4.3 LOCATION OF PROTEINS IN THE YEAST B
ACT
COMPLEX 100
4.3.1 CORRELATION OF THE 2D STRUCTURES OF THE YEAST B WITH THE B
ACT
COMPLEX 101
4.3.2 POSITION OF PRP2 IN RELATION TO THE U5-SPECIFIC PROTEINS 102
4.3.3 THE POSITION OF CWC2 AND ITS IMPLICATIONS FOR THE POSITION OF THE
NTC 103
4.3.4 THE LOCATION OF CWC2, PRP8 AND PRP2 CAN PROVIDE INFORMATION ABOUT
THE
POSITION OF THE CATALYTIC CENTER 105
5 REFERENCES 107
IV TABT.F. OF CONTENTS
6 APPENDIX 117
6.1 LIST OF ABBREVIATIONS 117
6.2 ACKNOWLEDGMENTS 120
6.3 (EIDESSTATTLICHE) VERSICHERUNGEN UND ERKLARUNGEN 123
|
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| spelling | Rigo, Norbert Verfasser (DE-588)1073305589 aut Electron microscopic location of tagged proteins in the yeast S. cerevisiae spliceosomal B and B act complexes vorgelegt von Norbert Rigo 2014 IV, 123 S. Ill., graph. Darst. txt rdacontent n rdamedia nc rdacarrier Bayreuth, Univ., Diss., 2014 Bierhefe (DE-588)4140270-4 gnd rswk-swf Elektronenmikroskopie (DE-588)4014327-2 gnd rswk-swf Spleißosom (DE-588)7724213-0 gnd rswk-swf Proteine (DE-588)4076388-2 gnd rswk-swf (DE-588)4113937-9 Hochschulschrift gnd-content Bierhefe (DE-588)4140270-4 s Spleißosom (DE-588)7724213-0 s Proteine (DE-588)4076388-2 s Elektronenmikroskopie (DE-588)4014327-2 s DE-604 DNB Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=028058193&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Rigo, Norbert Electron microscopic location of tagged proteins in the yeast S. cerevisiae spliceosomal B and B act complexes Bierhefe (DE-588)4140270-4 gnd Elektronenmikroskopie (DE-588)4014327-2 gnd Spleißosom (DE-588)7724213-0 gnd Proteine (DE-588)4076388-2 gnd |
| subject_GND | (DE-588)4140270-4 (DE-588)4014327-2 (DE-588)7724213-0 (DE-588)4076388-2 (DE-588)4113937-9 |
| title | Electron microscopic location of tagged proteins in the yeast S. cerevisiae spliceosomal B and B act complexes |
| title_auth | Electron microscopic location of tagged proteins in the yeast S. cerevisiae spliceosomal B and B act complexes |
| title_exact_search | Electron microscopic location of tagged proteins in the yeast S. cerevisiae spliceosomal B and B act complexes |
| title_full | Electron microscopic location of tagged proteins in the yeast S. cerevisiae spliceosomal B and B act complexes vorgelegt von Norbert Rigo |
| title_fullStr | Electron microscopic location of tagged proteins in the yeast S. cerevisiae spliceosomal B and B act complexes vorgelegt von Norbert Rigo |
| title_full_unstemmed | Electron microscopic location of tagged proteins in the yeast S. cerevisiae spliceosomal B and B act complexes vorgelegt von Norbert Rigo |
| title_short | Electron microscopic location of tagged proteins in the yeast S. cerevisiae spliceosomal B and B act complexes |
| title_sort | electron microscopic location of tagged proteins in the yeast s cerevisiae spliceosomal b and b act complexes |
| topic | Bierhefe (DE-588)4140270-4 gnd Elektronenmikroskopie (DE-588)4014327-2 gnd Spleißosom (DE-588)7724213-0 gnd Proteine (DE-588)4076388-2 gnd |
| topic_facet | Bierhefe Elektronenmikroskopie Spleißosom Proteine Hochschulschrift |
| url | http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=028058193&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
| work_keys_str_mv | AT rigonorbert electronmicroscopiclocationoftaggedproteinsintheyeastscerevisiaespliceosomalbandbactcomplexes |