Structural charaterization of the EF-G mediated translocation reaction: Appendix: pseudo-atomic model of the human 80S ribosome
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| Format: | Thesis Book |
| Language: | English |
| Published: |
Berlin
2013
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| Subjects: | |
| Online Access: | Inhaltsverzeichnis |
| Physical Description: | 116 S. Ill., graph. Darst. |
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| 245 | 1 | 0 | |a Structural charaterization of the EF-G mediated translocation reaction |b Appendix: pseudo-atomic model of the human 80S ribosome |c von David Ramrath |
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|---|---|
| adam_text | IMAGE 1
TABLE OF CONTENTS
SUMMARY 2
ZUSAMMENFASSUNG 4
1. INTRODUCTION
1.1 GENERAL INTRODUCTIONS ABOUT THE RIBOSOME AND TRANSLATION 7
1.2 RELEVANCE OF CRYO-ELECTRON MICROSCOPY FOR THE RIBOSOMAL RESEARCH 10
1.3 CONFORMATIONAL DYNAMICS OF THE RIBOSOME AND SINGLE MOLECULE
FLUORESCENCE
RESONANCE ENERGY TRANSFER (SMFRET) 11
1.4 TRANSLATIONAL GTPASES 13
1.5 PROTEIN BIOSYNTHESIS IN BACTERIA
1.5.1 INITIATION 14
1.5.2 THE ELONGATION CYCLE OF PROTEIN SYNTHESIS 15
1.5.3 TERMINATION AND RECYCLING 19
1.6 TRANS-TRANSLATION 19
1.7 SPECIFIC AIMS OF THE PRESENTED WORK 21
2. MATERIALS AND METHODS
2.1 SOURCES OF CHEMICALS, DEVICES AND SOFTWARE 23
2.2 BUFFERS, SOLUTION AND MEDIA COMPOSITIONS 26
2.3 BIOCHEMICAL METHODS
2.3.1 CONCENTRATION MEASUREMENT FOR NUCLEIC ACIDS 31
2.3.2 PROTEIN-SDS-POLYACRYLAMIDE GEL ELECTROPHORESIS 31
2.3.3 RNA/DNA GEL ELECTROPHORESIS 33
2.3.4 WESTERN BLOT AND IMMUNO-DETECTION OF PROTEINS 34
2.3.5 ANALYTICAL DENSITY GRADIENT CENTRIFUGATION 35
2.3.6 COLD TCA PRECIPITATION PROTEINS 35
2.3.7 ANALYTICAL SUCROSE CUSHION CENTRIFUGATION 36
2.3.8 POLY(U)-DEPENDENT POLY(PHE) SYNTHESIS 37
2.3.9 NON-ENZYMATIC SITE-SPECIFIC TRNA BINDING (WATANABE ASSAY) 37
2.3.10 ISOLATION OF TIGHT-COUPLED 70S FROM E. COLI 39
2.3.11 PREPARATIVE ISOLATION OF 30S AND 50S SUBUNITS 40
2.3.12 PREPARATION OF RE-ASSOCIATED 70S 40
2.3.13 PURIFICATION OF HETERO-POLYMERIC MESSENGER RNA 41
2.4 CRYO-EM AND COMPUTATIONAL METHODS
2.4.1 CRYO-EM GRID PREPARATION 41
2.4.2 CRYO-EM DATA COLLECTION 42
2.4.3 CRYO-EM DATA PROCESSING, MULTI-PARTICLE CLASSIFICATION AND
REFINEMENT 42
2.4.4 RNA-MODELING 43
3. RESULTS
3.1 GTP-HYDROLYSIS IN RIBOSOME BOUND EF-G 3.1.1 INFLUENCE OF A CHEMICAL
MODIFICATION TOWARDS THE RIBOSOMAL DYNAMICS 45
5
HTTP://D-NB.INFO/1041837356
IMAGE 2
TABLE OF CONTENTS
3.1.2 MULTI PARTICLE CLASSIFICATION OF THE 70S, (CY3-)TRNA FMET ,
EF-GGDPNP SAMPLE
3.1.3 THE EXTENDED SWITCH I REGION OF EF-G
3.1.4 THE INTERACTION BETWEEN THE H8/HL4-JUNCTION AND EF-G SWITCH I
3.2 DIRECT VISUALIZATION OF THE FA-STALLED TRANSLOCATION INTERMEDIATE
3.2.1 OVERVIEW OF THE CANONICAL TI POST COMPLEX
3.2.2 THE INTRA-SUBUNIT AP/P AND PE/E HYBRID SITES - A NOVEL PAIR OF
TRNA BINDING SITES
3.2.3 A NOVEL INTER-SUBUNIT BRIDGE BETWEEN THE ASF, L5 AND S19
3.2.4 THE ROLE OF EF-G DOMAIN IV DURING TRANSLOCATION
3.2.5 TENTATIVE ASSIGNMENT OF THE CONFIGURATION OF A1492 AND A1493
3.2.6 THE PE/E-TRNA AND ITS INTERACTION WITH THE MRNA
3.3 THE COMPLEX OF TMRNA*SMPB AND EF-G ON TRANSLOCATING RIBOSOMES
3.3.1 OVERVIEW OF THE TMRNA-TI POST COMPLEX
3.3.2 PSEUDO-ATOMIC MODEL OF FULL-LENGTH TMRNA
3.3.3 LIGAND CONFIGURATION AND OVERALL RIBOSOMAL CONFORMATION
3.3.4 CHARACTERIZATION OF THE TLD SMPB MODULE AND TRNA* 61 BINDING SITES
3.3.5 THE EF-G - SMPB INTERACTION AROUND THE 30S DECODING CENTER
3.4 PREPARATION OF A 70SEF-G COMPLEX CONTAINING AN A-TRNA (TI PRE )
3.4.1 IN VITRO PREPARATION OF THE CANONICAL TI PRE COMPLEX
3.4.2 MULTI PARTICLE CLASSIFICATION OF THE PRE RIBOSOMES, EF-G,
GDPNP AND VIOMYCIN PREPARATION
4. DISCUSSION
4.1 CANONICAL TRANSLOCATION 4.1.1 GTP-HYDROLYSIS IN EF-G
4.1.2 THE EF-G CATALYZED 30S-UNLOCKING
4.1.3 COUPLING OF BODY/PLATFORM BACK-ROTATION WITH
THE GTP-TO-GDP TRANSITION IN EF-G
4.1.4 THE REGULATION OF THE READING FRAME MAINTENANCE DURING
TRANSLOCATION
4.2 TRANSLOCATION DURING TRANS-TRANSLATION
4.2.1 COUPLING OF TMRNA SMPB TRANSLOCATION TO MLD-LOADING
4.2.2 TUNABLE AND VERSATILE DYNAMICS OF THE RIBOSOME
5. APPENDIX
5.1 MODEL OF THE 80S HUMAN RIBOSOME
6. REFERENCES
7. ATTACHMENTS
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| spelling | Ramrath, David 1981- Verfasser (DE-588)1037757416 aut Structural charaterization of the EF-G mediated translocation reaction Appendix: pseudo-atomic model of the human 80S ribosome von David Ramrath Berlin 2013 116 S. Ill., graph. Darst. txt rdacontent n rdamedia nc rdacarrier Berlin, Humboldt-Univ., Diss., 2013 (DE-588)4113937-9 Hochschulschrift gnd-content Reproduziert als Ramrath, David Structural charaterization of the EF-G mediated translocation reaction Appendix: pseudo-atomic model of the human 80S ribosome Ketsch bei Mannheim : Mikroform Dissertation, 2013 2 Mikrofiches (DE-604)BV041709942 DNB Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=026168144&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Ramrath, David 1981- Structural charaterization of the EF-G mediated translocation reaction Appendix: pseudo-atomic model of the human 80S ribosome |
| subject_GND | (DE-588)4113937-9 |
| title | Structural charaterization of the EF-G mediated translocation reaction Appendix: pseudo-atomic model of the human 80S ribosome |
| title_auth | Structural charaterization of the EF-G mediated translocation reaction Appendix: pseudo-atomic model of the human 80S ribosome |
| title_exact_search | Structural charaterization of the EF-G mediated translocation reaction Appendix: pseudo-atomic model of the human 80S ribosome |
| title_full | Structural charaterization of the EF-G mediated translocation reaction Appendix: pseudo-atomic model of the human 80S ribosome von David Ramrath |
| title_fullStr | Structural charaterization of the EF-G mediated translocation reaction Appendix: pseudo-atomic model of the human 80S ribosome von David Ramrath |
| title_full_unstemmed | Structural charaterization of the EF-G mediated translocation reaction Appendix: pseudo-atomic model of the human 80S ribosome von David Ramrath |
| title_short | Structural charaterization of the EF-G mediated translocation reaction |
| title_sort | structural charaterization of the ef g mediated translocation reaction appendix pseudo atomic model of the human 80s ribosome |
| title_sub | Appendix: pseudo-atomic model of the human 80S ribosome |
| topic_facet | Hochschulschrift |
| url | http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=026168144&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
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