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Structure-function relationships, tertiary interactions and thermostability of RNase P [Elektronische Ressource] / vorgelegt von Michal Marszalkowski

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131 Pages
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Structure-function relationships, tertiary interactions and thermostabilty of RNase P Dissertation zur Erlangung des Doktorgrades der Naturwissenschaften (Dr. rer. nat.) dem Fachbereich Pharmazeutische Chemie der Philipps-Universität Marburg vorgelegt von Michal Marszalkowski Marburg/Lahn 2007 Vom Fachbereich Pharmazeutische Chemie der Philipps-Universität Marburg als Dissertation am 14 November 2007 angenommen. Erstgutachter: Prof. Dr. Roland K. Hartmann Zweitgutachter: PD Dr. Klaus Reuter Tag der mündlichen Prüfung am: 14 November 2007 Table of Contents I Table of Contents I 1. Introduction 1 1.1. Ribozymes 1 1.2. RNase P 1 1.3. The RNA subunit of bacterial RNase P 3 1.4. Substrate recognition by RNase P 5 1.5. The catalytic mechanism of cleavage 5 1.6. The protein subunit of bacterial RNase P 7 1.7. Crystal structure, long range tertiary interactions and thermostability of bacterial RNase P RNA 8 1.8. References 14 2. Goal of the project 22 3. Methods 24 3.1. Bacterial cell culture 24 3.1.1. Cell growth on agar plates 25 3.1.2. Preparation of competent cells 25 3.1.2.1. Preparation of chemically competent E. coli cells, RbCl method 25 3.1.2.2. Preparation of electrocompetent E. coli cells 25 3.1.3. Transformation 26 3.1.3.1. Transformation of chemically competent E. coli cells 26 3.1.3.2.

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Published 01 January 2007
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Structure-function relationships, tertiary interactions
and thermostabilty of RNase P




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



dem Fachbereich
Pharmazeutische Chemie
der Philipps-Universität Marburg
vorgelegt von



Michal Marszalkowski





Marburg/Lahn 2007



























Vom Fachbereich Pharmazeutische Chemie
der Philipps-Universität Marburg als Dissertation am 14 November 2007
angenommen.

Erstgutachter: Prof. Dr. Roland K. Hartmann
Zweitgutachter: PD Dr. Klaus Reuter

Tag der mündlichen Prüfung am: 14 November 2007 Table of Contents I
Table of Contents I
1. Introduction 1
1.1. Ribozymes 1
1.2. RNase P 1
1.3. The RNA subunit of bacterial RNase P 3
1.4. Substrate recognition by RNase P 5
1.5. The catalytic mechanism of cleavage 5
1.6. The protein subunit of bacterial RNase P 7
1.7. Crystal structure, long range tertiary interactions and thermostability of bacterial
RNase P RNA 8
1.8. References 14
2. Goal of the project 22
3. Methods 24
3.1. Bacterial cell culture 24
3.1.1. Cell growth on agar plates 25
3.1.2. Preparation of competent cells 25
3.1.2.1. Preparation of chemically competent E. coli cells, RbCl method 25
3.1.2.2. Preparation of electrocompetent E. coli cells 25
3.1.3. Transformation 26
3.1.3.1. Transformation of chemically competent E. coli cells 26
3.1.3.2. Transformation of electrocompetent E. coli cells (electroporation) 26
3.1.4. In vivo complementation of E.coli strain BW 26
3.2. General nucleic acids techniques 27
3.2.1. Nucleic acid gel electrophoresis 27
3.2.1.1. Agarose gel electrophoresis 27
3.2.1.2. Crystal violet gels 28
3.2.1.3. Polyacrylamide gel electrophoresis (PAGE) 28
3.2.1.3.1. Denaturing PAGE 28
3.2.1.3.2. Native polyacrylamide gels 29
3.2.1.3.2.1. Non-denaturing polyacrylamide gel electrophoresis for RNA folding analysis
30
3.2.2. Detection of nucleic acids in gels 31
3.2.2.1. Ethidium bromide staining 31
3.2.2.2. UV-shadowing 31
3.2.2.3. Visualization using crystal violet 31
3.2.2.4. Radioluminography 31
3.2.3. Gel elution of nucleic acids 32
3.2.3.1. Elution by diffusion 32
3.2.4. Isolation of DNA from agarose gels 32
3.2.5. Photometric concentration determination of nucleic acids 32
3.2.6. Ethanol precipitation 33 Table of Contents II
3.2.7. Phenol/chloroform extraction 33
3.3. DNA techniques 33
3.3.1. Preparation of genomic DNA 33
3.3.2. Preparation of plasmid DNA 34
3.3.2.1. Preparative plasmid DNA isolation from E. coli cells 34
3.3.2.2. Analytical scale preparation of plasmid DNA (Mini prep) 34
3.3.3. Restriction digest of DNA 35
3.3.4. 5’- Phosphorylation of DNA 35
3.3.5. Ligation 36
3.3.6. Polymerase chain reaction (PCR) 36
3.4. RNA Techniques 38
3.4.1. Total RNA isolation 38
3.4.1.1. Trizol RNA isolation 38
3.4.1.2. Column RNA isolation 38
3.4.2. T7 Transcription of unmodified RNA 38
3.4.3. T7 Transcription of RNA carrying randomly distributed phosphorothioate analogues
40
323.4.4. 5’- end labelling of RNA with g- P ATP 41
323.4.5. 3’- end labelling of RNA with [5’- P] pCp 41
3.4.6. Folding analysis on non-denaturing gels 42
3.4.7. Partial hydrolysis of T. thermophilus P RNA by nuclease T1 43
3.4.8. Lead-induced hydrolysis of T. thermophilus P RNA 44
3.4.9. Iodine-induced hydrolysis of phosphorothioate analogue-modified RNA 44
3.4.10. Construction and analysis of a cDNA library from Hydrogenobacter thermophilus
TK6 45
3.4.10.1. Total RNA isolation, DNase I digestion and RNase P processing assay 46
3.4.10.2. Gel fractionation and RNase P processing assay 46
3.4.10.3. cDNA synthesis 47
3.4.10.3.1. C-tailing reaction 47
3.4.10.3.2. TAP treatment 48
3.4.10.3.3. 5’ RNA/DNA adapter ligation 48
3.4.10.3.4. First strand synthesis by reverse transcriptase 49
3.4.10.3.5. Second strand synthesis (PCR) 50
3.4.10.4. TA cloning and blue white screening 50
3.4.10.5. Colony PCR 50
3.4.10.6. In vitro transcription and RNase P processing assay 51
3.4.11. FPLC (Fast Performance Liquid Chromatography) 51
3.4.11.1. RNase P processing assays with FPLC fractions 52
3.4.12. Micrococcal nuclease treatment 52
3.5. Protein methods 53
3.5.1. Laemmli SDS-PAGE 53
3.5.2. Coomassie staining 54
3.5.3. Preparation of recombinant RNase P proteins 54
3.6. Kinetic Analysis 55
3.6.1. Kinetic analysis of in vitro reconstituted RNase P holoenzymes 55
3.6.2. Kinetic analysis of RNase P RNA 56
3.6.3. Evaluation of kinetic analysis 57
3.7. Cloning experiments 57 Table of Contents III
3.7.1. Plasmids for T7 transcriptions of diverse P RNAs 57
3.7.2. Construction of derivatives of the low copy plasmids pACYC177 for
complementation studies in E.coli rnpB mutant strains 58
3.8. References 60
4. Results and Discussion 61
4.1. RNase P in the Aquificales 61
4.1.1. References 67
4.2. Thermostabile RNase P RNAs lacking P18 identified in the Aquificales 68
4.3. Structural basis of a ribozyme’s thermostability: P1-L9 interdomain interaction in
RNase P RNA 78
4.4. In vitro and in vivo role of interdomain contacts in RNase P RNAs from
psychrophilic, mesophilic and thermophilic bacteria 94
5. Summary 107
6. Appendix 110
6.1. Chemicals 110
6.2. Radioisotopes 110
6.3. Size markers 111
6.4. Enzymes 111
6.5. Equipment 111
6.6. Synthetic DNA Oligonucleotides 112
6.7. Bacterial strains 116
6.8. Plasmid vectors 116
6.9. Plasmid vectors for T7 transcriptions 116
6.10. Abbreviations and Units 116
6.11. Index of Buffers and Solutions 118
6.12. Re ferences 119
7. Acknowledgements 120
8. Publications arising from this work 121
9. Curriculum vitae 122
10. Selbstständigkeitserklärung 123


Introduction 1
1. Introduction
1.1. Ribozymes
A ribozyme (from ribonucleic acid enzyme, also called RNA enzyme or catalytic RNA) is a
RNA molecule that catalyses a chemical reaction. Most natural ribozymes catalyse cleavage
of the molecule they are part of (cleavage in cis) but also the aminotransferase activity of the
ribosome was found to be essentially RNA-catalysed (activity in trans).
Before the discovery of ribozymes, protein enzymes were the only known biological catalysts.
The first ribozymes were discovered in the 1980s by Thomas R.Cech, who was studying RNA
splicing in the ciliated protozoan Tetrahymena thermophila, and by Sidney Altman, who was
working on bacterial ribonuclease P (RNase P) complexes. In 1989 T.R.Cech and S.Altman
won the Nobel Prize in chemistry for their “discovery of catalytic properties of RNA.” The
term ribozyme was first introduced by Kelly Kruger et al. in their 1982 Cell publication.
Nowadays, ribozymes are classified in terms of their diverse size and function. Naturally
existing catalytic RNAs include small ribozymes like the hammerhead, hairpin, hepatitis delta
virus and Varkud satellite ribozymes (Symons, 1992; Carola & Eckstein, 1999), and large
ribozymes like group I and II introns (Kruger at al., 1982) and the RNA subunit of RNase P
called P RNA (Guerrier-Takada et al., 1983). Further, among the ribozymes there exist two
major ribonucleoprotein machines, the ribosome (Nissen et al., 2000; Muth et al., 2000, Steitz
& Moore, 2003) and the spliceosome (Collins & Guthrie, 2000; Valadkhan & Manley, 2001).
All known ribozymes are metalloenzymes: for their catalytic activity and proper tertiary
2+ 2+folding they require divalent cations (Mg or Mn , Sun & Harris, 2007). The reaction
catalysed by ribozymes is an endonucleolytic transesterification of a phosphodiester bond
with an activated nucleophile. In contrast to all known ribozymes that act in cis, there is a
single naturally occurring one that cleaves RNAs in trans: RNase P (Tanner, 1999).
1.2. RNase P
Ribonuclease P (RNase P) is a ribonucleoprotein that is responsible for the 5’-maturation of
precursor tRNAs (pre-tRNAs). It is an essential ubiquitous endonuclease that is found in cells
from all three domains of life: the Bacteria , Eukarya and Archaea (Kazantsev & Pace, 2006;
Hartmann & Hartmann, 2003; Lee et al., 1991; Jarrous & Altman, 2001). RNase P carries out
a simple enzymatic reaction: the hydrolysis of a specific phosphodiester in pre-tRNAs to
release the 5’- flank and thereby generate tRNAs with mature 5’ends. E. coli RNase P also Introduction 2
cleaves other substrates, such as some viral RNAs (Mans et al., 1990; Hartmann et al., 1995),
p4.5S RNA (Peck-Miller & Altman, 1991), ptmRNA (Komine et al., 1994), a few mRNAs
(Li & Altman, 2003; Alifano et al., 1994) and some riboswitches (Altman et al., 2005).
RNase P is required for cell viability (Schedl & Primakoff, 1973, Gößringer et al., 2006). So
far, all known RNase P enzymes consist of one RNA subunit and at least one protein subunit
(Fig. 1.1) encoded by the rnpB and rnpA genes n bacteria. Both the RNA and protein
components are essential in vivo. An exception might be protein-only enzymes acting like
RNase P in spinach chloroplasts (Wang et al., 1988, Thomas et al., 1995) and mitochondria of
Trypanosoma brucei (Salavati et al., 2001).

Fig. 1.1: RNase P holoenzymes from Bacteria, Archaea and Eukarya. The RNA subunit is marked in blue. The
single protein subunit from bacteria is indicated by a purple oval. Archaeal proteins are shown in green and
eukaryal ones in green (homologs of archaeal P proteins) and gray. All RNAs share conserved nucleotides in
helix P4 (indicated in red). In Eukarya there is a sibling of RNase P, RNase MRP (dark green), which is involved
in processing of precursor rRNAs and shares the majority of its protein subunits with eukaryal RNase P. In
addition, the core of its RNA subunit is structurally related to eukaryal P RNA. (Figure from Willkomm &
Hartmann, 2007).
Catalytic activity in general seems to reside in the RNA subunit of RNase P (Guerrier-Takada
et al., 1983). Thus at high concentrations of divalent metal ions all bacterial RNase P RNAs
analysed so far display catalytic activity in the absence of the protein when tested in vitro.
RNA alone activity in vitro has also been demonstrated under very high ion concentrations for
some Archaea (Hall & Brown, 2002), and recently also residual activity for eukaryal RNase P
RNAs has been shown (Kikovska at al., 2007).