Structural studies of ribosome stalling and translocation complexes [Elektronische Ressource] / Birgit Seidelt

Birgit Seidelt

Informations

• Publié par: ludwig-maximilians-universitat_munchen
• Publié le: 01 janvier 2010
• Nombre de lectures: 39
• Langue: Deutsch
• Poids de l'ouvrage: 4 Mo

Extrait

Dissertation zur Erlangung des Doktorgrades
der Fakultät für Chemie und Pharmazie
der Ludwig-Maximilians-Universität München




Structural studies of ribosome stalling
and translocation complexes






Birgit Seidelt
aus
Strausberg

2010


Erklärung

Diese Dissertation wurde im Sinne von §13 Abs. 3 bzw. 4 der Promotionsordnung vom
29. Januar 1998 von Herrn Prof. Dr. Roland Beckmann betreut.



Ehrenwörtliche Versicherung

Diese Dissertation wurde selbständig, ohne unerlaubte Hilfe erarbeitet.


München, am……………..



…………………………………..
Birgit Seidelt








Dissertation eingereicht am 07.05.2010
1. Gutachter: Prof. Dr. Roland Beckmann
2. Gutachter: Prof. Dr. Karl-Peter Hopfner
Mündliche Prüfung am 05.07.2010
TABLE OF CONTENTS


TABLE OF CONTENTS

SUMMARY ............................................................................................................................... 1
I INTRODUCTION ................................................................................................................ 2
1.1 Cryo-electron microscopy and single particle reconstruction .......................................... 2
1.2 Structure and function of the prokaryotic ribosome ......................................................... 5
1.2.1 Protein synthesis ........................................................................................................ 6
1.2.2 The ribosomal peptidyl transferase center ................................................................. 7
1.2.3 The ribosomal peptide exit tunnel ............................................................................. 9
1.3 Translational stalling in prokaryotes .............................................................................. 11
1.3.1 Ribosome stalling on the TnaC nascent peptide ...................................................... 11
1.3.2 Ribosome stalling on the SecM nascent peptide ..................................................... 14
1.4 Prokaryotic membrane insertion pathways..................................................................... 17
1.4.1 The Sec pathway for membrane insertion ............................................................... 18
1.4.2 The YidC-only pathway .......................................................................................... 19
1.5 The YidC/Oxa1/Alb3 protein family .............................................................................. 20
1.5.1 Structure and function of Escherichia coli YidC .................................................... 21
1.5.2 Structure and function of Streptococcus mutans YidC ........................................... 22
1.6 Aims of the study ............................................................................................................ 23
II MATERIALS AND METHODS ..................................................................................... 24
2.1 Methods in molecular biology ........................................................................................ 24
2.1.1 Polymerase chain reaction ....................................................................................... 24
2.1.2 Restriction digestion of DNA .................................................................................. 26
2.1.3 Ligation of DNA fragments ..................................................................................... 26
2.1.4 Transformation of Escherichia coli cells................................................................. 26
2.1.5 Isolation of plasmid DNA and DNA Sequencing ................................................... 27
2.1.6 Agarose gel electrophoresis ..................................................................................... 27
2.2 Methods in protein biochemistry .................................................................................... 27
2.2.1 Preparation of TnaC-stalled ribosome nascent chain complexes (RNCs) ............... 27
2.2.2 Preparation of SecM-stalled DP120-RNCs ............................................................. 31
2.2.3 Puromycin assay ...................................................................................................... 33
2.2.4 Purification of Streptococcus mutans YidC2 .......................................................... 33
2.2.5 Reconstitution assays ............................................................................................... 35
2.2.6 Protein precipitation and SDS-PAGE...................................................................... 36
TABLE OF CONTENTS


2.2.7 Western blot analysis ............................................................................................... 37
2.2.8 Preparation of YidC2-proteoliposomes ................................................................... 37
2.2.9 Flotation binding assays .......................................................................................... 39
2.2.10 Dynamic Light Scattering ...................................................................................... 40
2.3 Electron microscopy ....................................................................................................... 40
2.3.1 Negative-stain electron microscopy ........................................................................ 40
2.3.2 Cryo-electron microscopy ....................................................................................... 41
2.4 Image processing and three-dimensional reconstruction................................................ 42
2.4.1 Power spectra and defocus determination ............................................................... 42
2.4.2 Particle selection ...................................................................................................... 42
2.4.3 Initial particle alignment .......................................................................................... 43
2.4.4 Three-dimensional reconstruction ........................................................................... 43
2.4.5 Refinement .............................................................................................................. 44
2.4.6 Three-dimensional reconstruction and sorting of the datasets ................................ 45
2.5 Structural interpretation and modeling ........................................................................... 45
III RESULTS ........................................................................................................................ 47
3.1 Structural insight into the TnaC stalling mechanism...................................................... 47
3.1.1 Purification of a TnaC-stalled 70S ribosome complex ............................................ 47
3.1.2 Cryo-EM structure of the stalled TnaC-70S ribosome complex ............................. 48
3.1.3 The TnaC nascent chain adopts an extended conformation .................................... 53
3.1.4 Nascent chain-ribosome interactions within the exit tunnel .................................... 54
3.1.5 Distinct conformations of nascent chains within the ribosomal exit tunnel ............ 57
3.1.6 Inactivation of the peptidyl transferase center ......................................................... 58
3.1.7 The decoding site on the 30S subunit of the TnaC-70S complex ........................... 60
3.2 Structural analysis of the SecM stalling mechanism ...................................................... 62
3.2.1 Purification of a SecM-stalled 70S ribosome complex ........................................... 62
3.2.2 Cryo-EM structure of the stalled SecM-70S ribosome complex ............................. 64
3.2.3 The decoding site of the SecM-70S cryo-EM reconstruction ................................. 64
3.2.4 The ribosomal exit tunnel of the SecM-70S cryo-EM reconstruction..................... 66
3.2.5 Purification of a SecM-stalled DP120-ribosome nascent chain complex ............... 68
3.2.6 Biochemical and structural analysis of the SecM-stalled DP120-RNCs ................. 70
3.3 Towards a high resolution structure of a ribosome-YidC complex ............................... 72
3.3.1 Purification of Streptococcus mutans YidC2 .......................................................... 72
3.3.2 Reconstitution and cryo-EM of the 70S ribosome-YidC2 complex ....................... 73
TABLE OF CONTENTS


3.3.3 Generation of YidC-substrate specific ribosome-nascent chain complexes ........... 75
3.3.4 Reconstitution and cryo-EM of a MscL-RNC-YidC complex ................................ 78
3.3.5 Reconstitution of the membrane protein YidC2 into proteoliposomes ................... 80
3.3.6 Reconstitution of a 70S ribosome-YidC2-proteoliposome complex ....................... 82
3.3.7 Attempts for downsizing of the liposomes and YidC2-proteoliposomes ................ 85
IV DISCUSSION .................................................................................