Insights into RNase P RNA structure and function by a retro-evolution approach [Elektronische Ressource] / vorgelegt von Dan Li

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• Publié par: philipps-universitat_marburg
• Publié le: 01 janvier 2009
• Nombre de lectures: 11
• Langue: English
• Poids de l'ouvrage: 6 Mo

Extrait

Insights into RNase P RNA structure and function by
a retro-evolution approach




Dissertation
zur
Erlangung des Doktorgrades
der Naturwissenschaften
(Dr. rer. nat.)



dem Fachbereich
Pharmazeutische Chemie
der Philipps-Universität Marburg
vorgelegt von



Dan Li
aus Guizhou, China




Marburg/Lahn 2009





























Vom Fachbereich Pharmazeutische Chemie
der Philipps-Universität Marburg als Dissertation am angenommen.

Erstgutachter: Prof. Dr. Roland K. Hartmann
Zweitgutachter: Prof. Dr. Albrecht Bindereif

Tag der mündlichen Prüfung am:



Table of Contents

1 Introduction ........................................................................................................................... 1

1.1 RNase P .......................................................................................................................... 1

1.2 The RNA subunit of RNase P ........................................................................................ 2
1.2.1 Bacterial RNase P RNA .......................................................................................... 2
1.2.2 Archaeal RNase P RNA 6
1.2.3 Eukaryal RNase P RNA 8

1.3 The RNase P protein subunit 10
1.3.1 The bacterial RNase P protein............................................................................... 10
1.3.2 The archaeal and eukaryal RNase P proteins........................................................ 11

1.4 Holoenzyme models of bacterial RNase P................................................................... 13

1.5 RNase P: an ideal natural model to study the transition from the RNA world to the
protein world ........................................................................................................................ 15

1.6 References .................................................................................................................... 17

2 Goal of the Project............................................................................................................... 23

3 Methods............................................................................................................................... 25

3.1 General nucleic acids techniques ................................................................................. 25
3.1.1 Isolation of plasmid DNA from bacteria.............................................................. 25
3.1.1.1 Growth of bacterial cultures........................................................................... 25
3.1.1.2 Isolation.......................................................................................................... 25
3.1.2 Gel electrophoresis................................................................................................ 26
3.1.2.1 Agarose gel electrophoresis 26
3.1.2.2 Polyacrylamide gel electrophoresis (PAGE).................................................. 27
3.1.3 Concentration determination 29
3.1.4 Polymerase chain reaction (PCR) ......................................................................... 30
3.1.4.1 Colony PCR.................................................................................................... 31
3.1.5 Ethanol precipitation ............................................................................................. 32
3.1.6 Phenol/ chloroform extraction............................................................................... 32

3.2 Cloning ......................................................................................................................... 33
3.2.1 Construction of recombinant plasmids.................................................................. 33
3.2.1.1 Restriction enzyme digest .............................................................................. 33
3.2.1.2 Vector preparation.......................................................................................... 33
3.2.1.3 Insert preparation............................................................................................ 34
3.2.1.4 DNA Ligation................................................................................................. 34
3.2.2 Plasmid mutants .................................................................................................... 35
3.2.2.1 Site-directed mutagenesis............................................................................... 35
3.2.2.2 Megaprimer mutagenesis 36
3.2.2.3 “Inside-out”-PCR mutagenesis ...................................................................... 37
3.2.3 Transformation...................................................................................................... 39
3.2.3.1 Preparation of chemically competent E. coli cells (DH5α) ........................... 39 3.2.3.2 Transformation of chemically competent E. coli cells (DH5α) ..................... 40
3.2.3.3 Selection of objective transformant................................................................ 40
3.2.4 TOPO cloning ....................................................................................................... 41
3.2.5 Plasmids generated throughout this study............................................................. 41
3.2.5.1 Plasmids for complementation assays............................................................ 42
3.2.5.2 Plasmids for RNA preparation ....................................................................... 49

3.3 Complementation assays .............................................................................................. 69
3.3.1 Complementation assays with DW2 strain ........................................................... 69
3.3.1.1 Preparation of electrocompetent E. coli cells (DW2) .................................... 69
3.3.1.2 Electroporation with electrocompetent E. coli cells (DW2) .......................... 69
3.3.1.3 Observation of phenotype (DW2) .................................................................. 70
3.3.2 Complementation assays with SSB318 strain....................................................... 70
3.3.2.1 Preparation of naturally competent B. subtilis cells (SSB318) ...................... 70
3.3.2.2 Transformation with naturally competent B. subtilis............ 72
3.3.2.3 Observation of phenotype (SSB318).............................................................. 72
3.3.3 Complementation assays with BW strain 72
3.3.3.1 Preparation of electrocompetent E. coli cells (BW)....................................... 73
3.3.3.2 Electroporation with electrocompetent E. coli cells (BW) ............................ 73
3.3.3.3 Observation of phenotype (BW) .................................................................... 73

3.4 RNA preparation .......................................................................................................... 74
3.4.1 Total RNA extraction............................................................................................ 74
3.4.2 In vitro run-off T7 transcription............................................................................ 74
3.4.2.1 Transcription .................................................................................................. 74
3.4.2.2 RNA purification with denaturing PAA gels ................................................. 76
3.4.2.3 RNA purification with Sephadex columns..................................................... 77
3.4.3 RNA with homogeneous 3’-ends .......................................................................... 78
3.4.4 RNA carrying randomly distributed phosphorothioate analogues........................ 79
3.4.5 Biotin-labeled RNA............................................................................................... 80

3.5 E. coli RNase P protein preparation ............................................................................. 81
3.5.1 Protein preparation ................................................................................................ 81
3.5.2 Methods used in protein preparation..................................................................... 83
3.5.2.1 SDS-PAGE..................................................................................................... 83
3.5.2.2 Dialysis........................................................................................................... 85
3.5.2.3 Concentration determination of protein ......................................................... 85
3.5.3 Quality assessment of RNase P protein................................................................. 86

3.6 Kinetic assays.... 87
323.6.1 5’-endlabeling of substrate with γ- P-ATP .......................................................... 88
3.6.2 P RNA alone kinetic assays ...............................................................