Translational regulation by specific mRNA protein interactions: studies in Saccharomyces cerevisiae and cell-free translation systems
Gespeichert in:
| 1. Verfasser: | |
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| Format: | Abschlussarbeit Buch |
| Sprache: | German |
| Veröffentlicht: |
1997
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| Schlagworte: | |
| Online-Zugang: | Inhaltsverzeichnis |
| Beschreibung: | [12], 113 Bl. Ill., graph. Darst. |
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Datensatz im Suchindex
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TABLE OF CONTENTS
ACKNOWLEDGEMENTS
TABLE OF CONTENTS
ABBREVIATIONS
SUMMARY
1. INTRODUCTION
1.1. GENERAL INTRODUCTION 1
1.2. PROTEIN SYNTHESIS IN EUKARYOTES 1
1.2.1. THE INITIATION PATHWAY 2
1.2.2. FEATURES OF THE MRNA INFLUENCING INITIATION 4
1.2.3. ALTERNATIVE INITIATION PATHWAYS 6
1.3. GLOBAL AND GENE SPECIFIC CONTROL OF PROTEIN SYNTHESIS 7
1.3.1. GLOBAL REGULATION OF TRANSLATION 7
1.3.2. REGULATION OF GCN4 MRNA TRANSLATION 9
1.3.3. REGULATION OF TRANSLATION BY SPECIFIC RNA/PROTEIN COMPLEXES
AT THE 5' UTR OF THE MRNA 10
1.3.4. REGULATION OF TRANSLATION BY SPECIFIC RNA/PROTEIN
COMPLEXES FORMING ELSEWHERE IN THE MRNA 12
1.3.5. CONTROL OF CELLULAR IRON METABOLISM 14
1.3.6. IRP-1 AND 2: REGULATION OF MRNA BINDING 16
1.3.7. THE MECHANISM OF TRANSLATIONAL REPRESSION BY IRP 19
1.3.8. A GENERAL MECHANISM OF TRANSLATIONAL REPRESSION BY
PROTEINS BINDING TO THE 5'UTR 21
1.3.9. EFFECT OF THE DISTANCE BETWEEN THE CAP STRUCTURE AND THE
BIBLIOGRAFISCHE INFORMATIONEN
HTTP://D-NB.INFO/951195484
IRE-IRP COMPLEX ON TRANSLATIONAL REGULATION 21
1.4. AIM OF THE THESIS 22
2. MATERIALS AND METHODS
2.1. BACTERIAL STRAINS, MEDIA AND GROWTH CONDITIONS 24
2.2. YEAST STRAINS, MEDIA AND GROWTH CONDITIONS 24
2.3.
E.COLI TRANSFORMATION AND PLASMID DNA PREPARATION 24
2.4. YEAST TRANSFORMATION 25
2.5. CONSTRUCTION OF RECOMBINANT PLASMIDS 26
2.6. EXPRESSION AND PURIFICATION OF RECOMBINANT PROTEINS 27
2.7. PREPARATION OF YEAST PROTEIN EXTRACTS 29
2.8. IN VITRO TRANSCRIPTION 29
2.9. WESTERN BLOTTING 30
2.10. CELL FREE TRANSLATIONS 30
2.11. EXTRACTION AND NORTHERN BLOT ANALYSIS OF RNA FROM
CELL-FREE TRANSLATION REACTIONS 31
2.12. GEL RETARDATION ASSAYS 32
2.13. UV CROSSLINKING ASSAY 33
2.14. SUCROSE GRADIENT ANALYSIS 33
2.15. FLUORESCENCE ACTIVATED CELL ANALYSIS AND SORTING OF YEAST CELLS 34
2.16. PHOSPHOR IMAGER QUANTITATIVE ANALYSIS 34
3. RESULTS
3.1. AN
IN VIVO
ASSAY FOR THE STUDY OF MRNA/PROTEIN INTERACTIONS
3.1.1. DESCRIPTION OF THE SYSTEM 35
3.1.2. SELECTION OF THE REPORTER GENE
35
3.1.3. SCREENING PROCEDURE 37
3.1.4. CHALLENGING THE SYSTEM WITH THE IRE/IRP-1 INTERACTION 39
3.1.5. FLUORESCENCE ACTIVATED CELL SORTING (FACS) OF THE CELLS WITH
REPRESSED GFP TRANSLATION 41
3.1.6. IMPROVEMENT OF FLUORESCENCE BY THE USE OF THE S65T GFP MUTANT 45
3.1.7. FLUORESCENCE ACTIVATED CELL SORTING (FACS) OF CELLS WITH
REPRESSED S65T-GFP TRANSLATION 47
3.1.8. DEFINITION OF THE REGION WITHIN THE FLUORESCENCE DISTRIBUTION
OF THE MIXED POPULATION THAT CONTAINS THE CELLS EXPRESSING THE
REPRESSOR PROTEIN 49
3.2. STUDY OF THE MECHANISTIC BASIS OF THE " IRE/IRP POSITION EFFECT"
3.2.1. THE IRE/IRP POSITION EFFECT 52
3.2.2. IRP-1 BINDING TO AN IRE LOCATED WITHIN THE OPEN READING FRAME 52
3.2.3. FORMATION OF 80S COMPLEXES ON ORF.CAT IN THE PRESENCE OF
IRP-1 53
3.2.4. EFFECT OF IRE/IRP-1 COMPLEXES ON THE ELONGATION PHASE OF
TRANSLATION 55
3.2.5. HOW DOES THE TRANSLATION INITIATION APPARATUS OVERCOME
CAP-DISTAL IRE/IRP COMPLEXES? 58
3.2.6. STABILITY OF IRE/IRP-1 COMPLEXES UNDER TRANSLATION CONDITIONS 60
3.2.7. THE IRE/IRP-1 POSITION EFFECT IN A WGE DERIVED CELL-FREE
TRANSLATION SYSTEM 64
3.3. CHARACTERISATION OF THE RNA-BINDING PROPERTIES OF THYMIDYLATE
SYNTHASE (TS) AND THE MECHANISM OF TS INDUCED CHANGES IN PROTEIN
SYNTHESIS.
3.3.1. BY WHICH MECHANISM CAN A PROTEIN BINDING TO THE OPEN READING
FRAME OF AN MRNA REGULATE TRANSLATION? 67
3.3.2. GENERATION OF RECOMBINANT HUMAN TS PROTEIN 67
3.3.3. RECOMBINANT HIS-HTS PROTEIN DOES NOT EXHIBIT SIGNIFICANT
RNA-BINDING ACTIVITY AND DOES NOT AFFECT TS MRNA TRANSLATION
IN A CELL-FREE SYSTEM 69
3.3.4. BINDING ACTIVITY OF RECOMBINANT
E.COLI TS 70
3.3.5. RECOMBINANT
E.COLI TS PROTEIN BINDS TO SEVERAL MRNAS IN VITRO 72
3.3.6. ADDITION OF R
E.COLI TS CAUSES A GENERAL REDUCTION OF PROTEIN
BIOSYNTHESIS IN RRL CELL-FREE SYSTEM 73
3.3.7. THE PREPARATION OF R
E.COLI TS CAUSES DEGRADATION OF TS AND CAT
MRNAS IN A TRANSLATION INDEPENDENT WAY 74
3.3.8. PURIFICATION OF THE RNA-BINDING AND RNA-DEGRADING ACTIVITIES
OF THE RE.COLI TS PREPARATION 77
3.3.9. EFFECT OF THE GEL FILTRATION COLUMN FRACTIONS ON MRNA
TRANSLATION AND INTEGRITY 78
4. DISCUSSION
4.1. STUDY OF REGULATION OF TRANSLATION BY SPECIFIC MRNA/PROTEIN
INTERACTIONS 81
4.2.
SACCHAROMYCES CEREVISIAE AS AN IN VIVO SYSTEM FOR THE
IDENTIFICATION AND CHARACTERIZATION OF RNA/PROTEIN INTERACTIONS 81
4.3. STUDY OF THE MECHANISTIC BASIS OF THE "POSITION EFFECT" 86
4.3.1. THE EFFECT OF AN IRE/IRP-1 COMPLEX FORMATION WITHIN THE OPEN
READING FRAME ON MRNA TRANSLATION 86
4.3.2. MECHANISM FOR THE PROGRESSION OF THE TRANSLATION INITIATION
MACHINERY PAST A CAP-DISTANT IRE/IRP-1 BARRIER 87
4.4.
THE RNA-BINDING CHARACTERISTICS OF RECOMBINANT HUMAN AND
E.COLI
THYMIDYLATE SYNTHASE
91
4.5. SYNOPSIS: REGULATION OF TRANSLATION BY MRNA/PROTEIN
INTERACTIONS: MODELS AND APPLICATIONS 94
5. REFERENCES 97 |
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| author | Paraskeva, Efrosyni |
| author_GND | (DE-588)11560460X |
| author_facet | Paraskeva, Efrosyni |
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| indexdate | 2024-10-09T16:07:31Z |
| institution | BVB |
| language | German |
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| physical | [12], 113 Bl. Ill., graph. Darst. |
| publishDate | 1997 |
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| spelling | Paraskeva, Efrosyni Verfasser (DE-588)11560460X aut Translational regulation by specific mRNA protein interactions studies in Saccharomyces cerevisiae and cell-free translation systems vorgelegt von Efrosyni Paraskeva 1997 [12], 113 Bl. Ill., graph. Darst. txt rdacontent n rdamedia nc rdacarrier Heidelberg, Univ., Diss., 1997 RNS-Bindungsproteine (DE-588)4178253-7 gnd rswk-swf In vitro (DE-588)4250701-7 gnd rswk-swf Translationskontrolle (DE-588)4379556-0 gnd rswk-swf Saccharomyces cerevisiae (DE-588)4178812-6 gnd rswk-swf (DE-588)4113937-9 Hochschulschrift gnd-content Saccharomyces cerevisiae (DE-588)4178812-6 s Translationskontrolle (DE-588)4379556-0 s RNS-Bindungsproteine (DE-588)4178253-7 s DE-604 In vitro (DE-588)4250701-7 s DNB Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=007818450&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Paraskeva, Efrosyni Translational regulation by specific mRNA protein interactions studies in Saccharomyces cerevisiae and cell-free translation systems RNS-Bindungsproteine (DE-588)4178253-7 gnd In vitro (DE-588)4250701-7 gnd Translationskontrolle (DE-588)4379556-0 gnd Saccharomyces cerevisiae (DE-588)4178812-6 gnd |
| subject_GND | (DE-588)4178253-7 (DE-588)4250701-7 (DE-588)4379556-0 (DE-588)4178812-6 (DE-588)4113937-9 |
| title | Translational regulation by specific mRNA protein interactions studies in Saccharomyces cerevisiae and cell-free translation systems |
| title_auth | Translational regulation by specific mRNA protein interactions studies in Saccharomyces cerevisiae and cell-free translation systems |
| title_exact_search | Translational regulation by specific mRNA protein interactions studies in Saccharomyces cerevisiae and cell-free translation systems |
| title_full | Translational regulation by specific mRNA protein interactions studies in Saccharomyces cerevisiae and cell-free translation systems vorgelegt von Efrosyni Paraskeva |
| title_fullStr | Translational regulation by specific mRNA protein interactions studies in Saccharomyces cerevisiae and cell-free translation systems vorgelegt von Efrosyni Paraskeva |
| title_full_unstemmed | Translational regulation by specific mRNA protein interactions studies in Saccharomyces cerevisiae and cell-free translation systems vorgelegt von Efrosyni Paraskeva |
| title_short | Translational regulation by specific mRNA protein interactions |
| title_sort | translational regulation by specific mrna protein interactions studies in saccharomyces cerevisiae and cell free translation systems |
| title_sub | studies in Saccharomyces cerevisiae and cell-free translation systems |
| topic | RNS-Bindungsproteine (DE-588)4178253-7 gnd In vitro (DE-588)4250701-7 gnd Translationskontrolle (DE-588)4379556-0 gnd Saccharomyces cerevisiae (DE-588)4178812-6 gnd |
| topic_facet | RNS-Bindungsproteine In vitro Translationskontrolle Saccharomyces cerevisiae Hochschulschrift |
| url | http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=007818450&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
| work_keys_str_mv | AT paraskevaefrosyni translationalregulationbyspecificmrnaproteininteractionsstudiesinsaccharomycescerevisiaeandcellfreetranslationsystems |