Analysis of the relationship between ribosomal protein and SSU processome assembly in Saccharomyces cerevisiae [Elektronische Ressource] / vorgelegt von Steffen Jakob

Sujets

• Biologie

Informations

• Publié par: universitat_regensburg
• Publié le: 01 janvier 2010
• Nombre de lectures: 20
• Langue: Deutsch
• Poids de l'ouvrage: 7 Mo

Extrait

Analysis of the relationship between ribosomal
protein and SSU processome assembly in
Saccharomyces cerevisiae







Dissertation
zur Erlangung des Doktorgrades der Naturwissenschaften (Dr. rer. nat.)
der naturwissenschaftlichen Fakultät III – Biologie und vorklinische Medizin -
der Universität Regensburg




vorgelegt von
Steffen Jakob
aus Wolfen
Januar 2010


























Promotionsgesuch eingereicht am: 13. Januar 2010

Die Arbeit wurde angeleitet von: Prof. Dr. Herbert Tschochner


Prüfungsausschuss:
Vorsitzender: Prof. Dr. Armin Kurtz
1. Prüfer: Prof. Dr. Herbert Tschochner
2. Prüfer: Prof. Dr. Rainer Deutzmann
3. Prüfer: Prof. Dr. Wolfgang Seufert


Tag der mündlichen Prüfung: 24. März 2010






Die vorliegende Arbeit wurde in der Zeit von April 2006 bis Januar 2010 am Lehrstuhl
Biochemie III des Institutes für Biochemie, Genetik und Mikrobiologie der
Naturwissenschaftlichen Fakultät III der Universität zu Regensburg unter Anleitung von Dr.
Philipp Milkereit im Labor von Prof. Dr. Herbert Tschochner angefertigt.















Ich erkläre hiermit, dass ich diese Arbeit selbst verfasst und keine anderen als die
angegebenen Quellen und Hilfsmittel verwendet habe.
Diese Arbeit war bisher noch nicht Bestandteil eines Prüfungsverfahrens.
Andere Promotionsversuche wurden nicht unternommen.



Regensburg, den 13. Januar 2010




Steffen Jakob
Table of Contents
Table of Contents
1 SUMMARY ...................................................................................................... 1
2 INTRODUCTION ............................................................................................. 3
2.1 The ribosome................................................................................................................. 3
2.1.1 Components of the ribosome ........................................................................................................................ 3
2.1.2 Ribosome Structure............................................................................................................................................ 3
2.2 Ribosome Biogenesis in S. cerevisiae........................................................................... 6
2.3 R-protein assembly in prokaryotes 11
2.3.1 In vitro assembly ................................................................................................................................................11
2.3.2 In vivo assembly...........................15
2.4 R-protein assembly in eukaryotes ............................................................................. 16
2.4.1 In vitro assembly..........................16
2.4.2 In vivo assembly...........................16
2.5 SSU processome / 90S pre-ribosome......................................................................... 18
2.6 Objective ..................................................................................................................... 23
3 RESULTS ....................................................................................................... 25
3.1 Construction and analysis of yeast strains conditionally expressing rpS4,
rpS21, rpS22 and rpS29.............................................................................................. 25
3.1.1 Strain construction ...........................................................................................................................................25
3.1.2 Analysis of rRNA processing phenotypes in conditional mutants of rpS4, rpS21, rpS22
and rpS29 .............................................................................................................................................................27
3.2 Assembly of the SSU-processome component Noc4p with pre-ribosomal
particles after blockage of SSU rRNA 5’, 3’ and central domain assembly............. 30
3.3 Assembly of other SSU processome components with pre-ribosomal particles
after blockage of SSU rRNA 5’, 3’ and central domain assembly ............................ 34
3.4 Semi Quantitative analysis of protein complexes using iTRAQ .............................. 37
3.4.1 Introduction ........................................................................................................................................................37
3.4.2 Characterisation of general features of the quantitation using iTRAQ .........................................40
3.4.3 Establishment of an assay for quantitative analysis of protein complexes using iTRAQ ........42
3.4.4 Comparative analysis of proteins co-purifying with Noc4p-TAP after in vivo depletion
of primary (rpS5) or secondary (rpS15) SSU rRNA 3’ domain binding proteins..........................44
3.4.5 Comparative analysis of the protein composition of early pre-ribosomes purified by
Utp4p-TAP after disruption of SSU 5’, 3’ or central domain assembly...........................................48
3.5 Assembly of ribosomal proteins in the temperature sensitive noc4-8 strain ........ 53
ITable of Contents
3.6 Assembly of ribosomal proteins in other temperature sensitive mutants of
NOC4 ............................................................................................................................ 58
3.7 Assembly of ribosomal proteins in the temperature sensitive nop14-2 strain .... 61
3.8 Characterisation of the temperature sensitive noc4-8 strain.................................. 63
3.9 Assembly of SSU processome components in the absence of Noc4p..................... 65
4 DISCUSSION ................................................................................................. 69
4.1 rRNA processing phenotypes of ribosomal proteins S4, S21, S22, and S29 and
correlation with their localisation in the SSU ........................................................... 69
4.2 Establishment of an assay for semiquantitative analysis of pre-ribosomal
particles ....................................................................................................................... 70
4.2.1 Affinity purification of pre-ribosomes: technical considerations ....................................................70
4.2.2 Characterisation of the protein composition of affinity-purified early pre-ribosomal
particles: identification of LSU biogenesis factors in early pre-ribosomes...................................71
4.2.3 Semiquantitative comparison of the protein composition of affinity purified pre-
ribosomes by iTRAQ and MALDI-MS/MS: general considerations ..................................................73
4.3 SSU rRNA 3’ domain assembly events and recruitment of SSU processome
components................................................................................................................. 73
4.4 SSU rRNA central domain assembly and recruitment of SSU processome
components 76
4.5 Noc4p is required for efficient assembly of the SSU 3’ (head) domain, but not
for assembly of UTP-A, UTP-B, UTP-C and Mpp10 SSU processome modules to
early pre-ribosomes.................................................................................................... 78
4.6 Ribosome biogenesis factors function in r-protein assembly – A common
theme? ......................................................................................................................... 80
5 MATERIALS AND METHODS........................................................................ 84
5.1 Materials ...................................................................................................................... 84
5.1.1 Chemicals.......................................84
5.1.2 Buffers and media .............................................................................................................................................84
5.1.3 Saccharomyces cerevisiae strains .................................................................................................................87
5.1.4 Plasmids................................................................................................................................................................93
5.1.5 Oligonucleotides...............................................................................................................................................95
5.1.6 Probes....................................................................................................................................................................98
5.1.7 Enzymes................................................................................................................................................................98
5.1.8 Kits ..........................................................................................................................................................................99
5.1.9 Siz