Conformational dynamics of the ribosomal decoding complex in bacteria: stimulation of the GTDase activity of elongation factor Tu
Gespeichert in:
| 1. Verfasser: | |
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| Format: | Abschlussarbeit Buch |
| Sprache: | English |
| Veröffentlicht: |
2013
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| Schlagworte: | |
| Online-Zugang: | Inhaltsverzeichnis |
| Beschreibung: | 190 S. Ill., graph. Darst. |
Internformat
MARC
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| 100 | 1 | |a Schütte, Jan-Christian |d 1980- |e Verfasser |0 (DE-588)1036380807 |4 aut | |
| 245 | 1 | 0 | |a Conformational dynamics of the ribosomal decoding complex in bacteria |b stimulation of the GTDase activity of elongation factor Tu |c von Jan-Christian Schütte |
| 264 | 1 | |c 2013 | |
| 300 | |a 190 S. |b Ill., graph. Darst. | ||
| 336 | |b txt |2 rdacontent | ||
| 337 | |b n |2 rdamedia | ||
| 338 | |b nc |2 rdacarrier | ||
| 502 | |a Berlin, Humboldt-Univ., Diss., 2013 | ||
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| 999 | |a oai:aleph.bib-bvb.de:BVB01-026079863 | ||
Datensatz im Suchindex
| _version_ | 1804150486499065856 |
|---|---|
| adam_text | IMAGE 1
CONTENTS
1 INTRODUCTION 13
1.1 THE STRUCTURE OF THE BACTERIAL RIBOSOME 13
1.2 BACTERIAL PROTEIN SYNTHESIS 16
1.2.1 INITIATION 17
1.2.2 ELONGATION CYCLE 19
1.2.3 RECODING OF THE STOP CODON UGA DURING SELENOCYSTEINE INCORPORATION
20
1.2.4 TERMINATION AND RELEASE OF THE NASCENT CHAIN 23
1.2.5 RECYCLING 24
1.2.6 ENERGY-CONSUMPTION OF PROTEIN BIOSYNTHESIS 24
1.3 STRUCTURAL DYNAMICS OF THE RIBOSOME 25
1.3.1 FLEXIBLE ELEMENTS OF THE RIBOSOME 26
1.3.2 THE ROTATION OF THE SMALL SUBUNIT AGAINST THE LARGE SUBUNIT ....
26
1.3.3 THE LI STALK REGION 27
1.3.4 THE L7/L12 STALK REGION AND THE GTPASE ASSOCIATED CENTRE .... 29
1.3.5 THE DECODING CENTER 29
1.3.6 THE PEPTIDYL-TRANSFERASE CENTER 31
1.3.7 TRNA BINDING SITES: THE THREE-SITE AND HYBRID-SITE MODELS .... 32
1.4 GTPASES 35
1.4.1 FUNCTION AND STRUCTURE OF ELONGATION FACTOR TU 3-5
1.4.2 FUNCTION AND STRUCTURE OF SELENOCCYSTEINE INCORPORATION FACTOR
SELB 38
1.5 STUDYING CONFORMATIONAL MODES OF RIBOSOMES WITH CRYO-EM 40
1.6 AIM OF THIS STUDY - INVESTIGATION OF STRUCTURAL DYNAMICS DURING
DECODING . 41
2 MATERIALS AND METHODS 43
2.1 BIOCHEMICAL PREPARATION OF RIBOSOMAL COMPLEXES 43
2.1.1 BUFFERS AND SOLUTIONS 43
HTTP://D-NB.INFO/1042804907
IMAGE 2
CONTENTS
2.1.2 RNA GELS 44
2.1.3 PURIFICATION OF RNA PRODUCTS 44
2.1.4 TERNARY COMPLEX FORMATION ASSAY 46
2.1.5 GTPASE ASSAY 47
2.1.6 MRNA DESIGN 48
2.1.7 PRIMER DESIGN FOR RECLONING 48
2.2 PREPARATION OF RIBOSOMAL SAMPLES FOR CRYO-EM 49
2.2.1 PREPARATION OF GRIDS FOR ELECTRON MICROSCOPY 49
2.2.2 VITRIFICATION OF RIBOSOME SAMPLES 50
2.3 CRYO-EM 51
2.3.1 BASIC PRINCIPLES OF ELECTRON MICROSCOPY 51
2.3.2 PHASE CONTRAST TRANSFER CORRECTION 52
2.3.3 IMAGING CONDITIONS FOR DATA COLLECTION 55
2.4 IMAGE DATA PROCESSING AND PARTICLE IMAGE SELECTION 56
2.4.1 DIGITALIZATION OF IMAGES 56
2.4.2 THE SPIDER SOFTWARE PACKAGE 56
2.4.3 THE SPARX SOFTWARE PACKAGE 57
2.4.4 EVALUATION OF POWER SPECTRA 57
2.4.5 AUTOMATIC DETERMINATION OF MICROGRAPH QUALITY 58
2.4.6 CALCULATION OF DEFOCUS VALUES 60
2.4.7 CONVERSION OF TIF FILES INTO SPIDER IMAGE FORMAT 61
2.4.8 PARTICLE SELECTION 62
2.5 3D RECONSTRUCTION: THE SINGLE-PARTICLE APPROACH 70
2.5.1 PROJECTION MATCHING OF PARTICLE IMAGES TO REFERENCE PROJECTIONS .
. 71
2.5.2 THE SIGNAL-TO-NOISE RATIO 73
2.5.3 IMPLEMENTATION OF SPIDER SCRIPTS ON UNIX-CLUSTER-SYSTEMS ... 73
2.5.4 LOW-PASS FILTRATION IN FOURIER SPACE 74
2.5.5 ADJUSTMENT OF THE DEFOCUS OF EM MAPS 74
2.5.6 REFINEMENT SCHEME AND PROGRESSION TO LOWER PIXEL SIZES 76
2.5.7 SPIDER ALGORITHMS USED IN THE REFINEMENT PROCESS 78
2.6 HIGH-RESOLUTION IMAGE PROCESSING 79
2.6.1 REEVALUATION OF DEFOCUS VALUES BY CROSS-RESOLUTION 80
2.6.2 COMBINATION OF DEFOCUS GROUPS 80
IMAGE 3
CONTENTS
2.6.3 APPLICATION OF A LAYERED MASK 81
2.6.4 HIGH-FREQUENCY ENHANCEMENT 81
2.7 MULTI-PARTICLE REFINEMENT 84
2.8 INTERPRETATION OF EM MAPS 86
2.8.1 RESOLUTION OF EM MAPS 86
2.8.2 MOLECULAR MODELING AND INTERPRETATION 87
2.8.3 LOCAL CROSS-CORRELATION MAPS 87
2.9 PROTEIN AND EM DATA BANK ENTRIES FOR THE T. THERMOPHILUS
SUBCONFORMA- TION I 88
3 RESULTS 89
3.1 NON-CANONICAL DECODING DURING SELENOCYSTEINE INCORPORATION 89
3.2 CANONICAL DECODING IN E. COLI 94
3.2.1 PREPARATION OF THE E. COLI DECODING COMPLEX 94
3.2.2 DATA PROCESSING OF THE DECODING COMPLEX IN E. COLI 94
3.2.3 THE STRUCTURE OF THE DECODING COMPLEX IN E. COLI 95
3.2.4 COMPOSITION OF THE DECODING COMPLEX IN E. COLI 97
3.3 CANONICAL DECODING IN T. THERMOPHILUS 100
3.3.1 PREPARATION OF THE T. THERMOPHILUS DECODING COMPLEX 100
3.3.2 INTRINSIC CONFORMATIONAL HETEROGENEITY OF THE T. THERMOPHILUS DE
CODING COMPLEX 102
3.4 EXTENSION OF THE DATA SET 104
3.5 SUBCONFORMATION I: THE NON-ROTATED STATE 105
3.5.1 DOCKING OF MOLECULAR MODELS INTO THE CRYO-EM MAP 105
3.5.2 EVALUATION OF THE ACCURACY OF THE DOCKED MODELS 108
3.5.3 ASSESSMENT OF THE QUALITY OF THE STRUCTURE 110
3.5.4 IDENTIFICATION OF CONTACTS BETWEEN THE LIGANDS AND THE RIBOSOME .
. 112
3.5.5 THE CONFORMATION OF THE A/T-TRNA 116
3.5.6 THE CONFORMATION OF EF-TU.GDP.KIRROMYCIN 120
3.6 SUBCONFORMATION II: THE ROTATED STATE 127
3.7 SUBCONFORMATION III: ARC-LIKE CONNECTION BETWEEN THE L7/L12 STALK
BASE
AND EF-TU 130
3.8 COMPARISON OF THE E.COLI WITH THE T. THERMOPHILUS STRUCTURES 138
IMAGE 4
CONTENTS
4 CONCLUSIONS 143
4.1 BIOCHEMICAL WORK 143
4.1.1 PROTEIN EXPRESSION OF THE SELB-EF-TU HYBRID PROTEIN IS DIFFICULT
IN
E. COLI 143
4.2 DATA PROCESSING OF PARTICLE IMAGES 144
4.2.1 AUTOMATED PARTICLE IMAGE SELECTION ALLOWS FOR ENLARGED DATA SETS .
144
4.2.2 AUTOMATED DETERMINATION OF MICROGRAPH QUALITY IS NOT RELIABLE FOR
LARGE, NOISY DATA SETS 145
4.3 CONFORMATIONAL DYNAMICS OF THE BACTERIAL RIBOSOME DURING DECODING .
. . 145
4.3.1 SEVERAL SUBCONFORMATIONS CAN BE ASSUMED IN THE DECODING STATE . .
145
4.3.2 THE METASTABLE FREE-ENERGY LANDSCAPE MODEL OF RIBOSOME FUNCTION .
147
4.3.3 THE ARC-LIKE CONNECTIONS DIFFER BETWEEN THE TWO SPECIES 148
4.4 CANONICAL DECODING IN T. THERMOPHILUS 151
4.4.1 THE CRYO-EM MAP OF THE T. THERMOPHILUS MAIN SUBCONFORMATION
ALLOWS FOR MOLECULAR INSIGHTS INTO THE CANONICAL DECODING PROCESS . 151
4.4.2 A COMPLEX DISTORTION OF THE TRNA TAKES PLACE IN THE A/T-STATE . .
152
4.4.3 RIBOSOMAL INTERACTIONS WITH THE TERNARY COMPLEX AND THEIR ROLE IN
GTPASE ACTIVATION 153
4.4.4 SUBCONFORMATION I RESEMBLES A METHYLKIRROMYCIN-STALLED COMPLEX .
155
4.4.5 THE SRL PLAYS AN ACTIVE ROLE IN THE ACTIVATION OF GTP-HYDROLYSIS
BY EF-TU 156
4.4.6 THE SWITCH I REGION OF EF-TU IS FRAGMENTED, HINTING AT A CONFOR
MATIONAL CHANGE 157
4.4.7 THE HYDROPHOBIC GATE APPEARS TO BE OPENED 158
4.4.8 A MODEL OF GTPASE ACTIVATION IN EF-TU DURING DECODING 158
4.5 COMPARISON OF THE MAIN CONFORMATION WITH OTHER HIGH RESOLUTION
STRUCTURES 160
4.6 SUMMARY AND OUTLOOK 164
APPENDIX 167
LIST OF PUBLICATIONS BY JAN-CHRISTIAN SCHUETTE 169
ABBREVIATIONS 171
ACKNOWLEDGEMENTS 172
LIST OF FIGURES 172
LIST OF TABLES 175
IMAGE 5
CONTENTS
SELBSTANDIGKEITSERKLARUNG 177
BIBLIOGRAPHY 180
|
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| author_facet | Schütte, Jan-Christian 1980- |
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| bvnumber | BV041103518 |
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| dewey-ones | 571 - Physiology & related subjects |
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| dewey-search | 571.6582932 |
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| dewey-tens | 570 - Biology |
| discipline | Biologie |
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| spelling | Schütte, Jan-Christian 1980- Verfasser (DE-588)1036380807 aut Conformational dynamics of the ribosomal decoding complex in bacteria stimulation of the GTDase activity of elongation factor Tu von Jan-Christian Schütte 2013 190 S. Ill., graph. Darst. txt rdacontent n rdamedia nc rdacarrier Berlin, Humboldt-Univ., Diss., 2013 (DE-588)4113937-9 Hochschulschrift gnd-content DNB Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=026079863&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis |
| spellingShingle | Schütte, Jan-Christian 1980- Conformational dynamics of the ribosomal decoding complex in bacteria stimulation of the GTDase activity of elongation factor Tu |
| subject_GND | (DE-588)4113937-9 |
| title | Conformational dynamics of the ribosomal decoding complex in bacteria stimulation of the GTDase activity of elongation factor Tu |
| title_auth | Conformational dynamics of the ribosomal decoding complex in bacteria stimulation of the GTDase activity of elongation factor Tu |
| title_exact_search | Conformational dynamics of the ribosomal decoding complex in bacteria stimulation of the GTDase activity of elongation factor Tu |
| title_full | Conformational dynamics of the ribosomal decoding complex in bacteria stimulation of the GTDase activity of elongation factor Tu von Jan-Christian Schütte |
| title_fullStr | Conformational dynamics of the ribosomal decoding complex in bacteria stimulation of the GTDase activity of elongation factor Tu von Jan-Christian Schütte |
| title_full_unstemmed | Conformational dynamics of the ribosomal decoding complex in bacteria stimulation of the GTDase activity of elongation factor Tu von Jan-Christian Schütte |
| title_short | Conformational dynamics of the ribosomal decoding complex in bacteria |
| title_sort | conformational dynamics of the ribosomal decoding complex in bacteria stimulation of the gtdase activity of elongation factor tu |
| title_sub | stimulation of the GTDase activity of elongation factor Tu |
| topic_facet | Hochschulschrift |
| url | http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=026079863&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
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