Analysis of components of the mitochondrial transcription machinery in Arabidopsis thaliana [Elektronische Ressource] / von Kristina Kühn

Tina Masson

Sujets

• Biologie

Informations

• Publié par: humboldt-universitat_zu_berlin
• Publié le: 01 janvier 2006
• Nombre de lectures: 20
• Langue: Deutsch
• Poids de l'ouvrage: 6 Mo

Extrait

Analysis of components
of the mitochondrial transcription machinery in
Arabidopsis thaliana

DISSERTATION
zur Erlangung des akademischen Grades
doctor rerum naturalium (Dr. rer. nat.) im Fach Biologie

eingereicht an der
Mathematisch-Naturwissenschaftlichen Fakultät I
der Humboldt-Universität zu Berlin

von
Diplom-Biologin Kristina Kühn
geboren am 27.08.1974 in Stollberg
Präsident der Humboldt-Universität zu Berlin in Vertretung
Prof. Dr. Hans Jürgen Prömel

Dekan der Mathematisch-Naturwissenschaftlichen Fakultät I
Prof. Thomas Buckhout, PhD

Gutachter: 1. Prof. Dr. Thomas Börner
2. Prof. Dr. Wolfgang Hess
3. Prof. Dr. Frank Kempken

Datum der mündlichen Prüfung: 24. Februar 2006

Abstract
In der vorliegenden Arbeit wurde die Transkription mitochondrialer Gene durch die
kernkodierten Phagentyp-RNA-Polymerasen RpoTm und RpoTmp der Pflanze Arabidopsis
untersucht.
Im Mitochondriengenom von Arabidopsis wurden für 12 Gene Promotoren bestimmt. Diese
zeigten verschiedene Sequenzelemente und wichen meist von der für Dikotyle publizierten
Konsensussequenz ab. Für die Mehrheit der Gene wurden multiple Promotoren identifiziert. Es
wurden weiterhin Promotoren nachgewiesen, welche die Transkription vermutlich nicht
funktioneller Sequenzen aktivieren. Architektur, Lokalisation und Nutzung mitochondrialer
Promotoren implizieren eine wenig stringente Kontrolle der Transkriptionsinitiation in
Arabidopsis-Mitochondrien.
Zur Analyse der Funktionen von RpoTm und RpoTmp wurde ein in vitro-Transkriptions-
system entwickelt. Da RpoT-Enzyme möglicherweise Kofaktoren benötigen, wurde in
Arabidopsis nach Genen potentieller mitochondrialer Transkriptionsfaktoren gesucht. Als
mitochondriales Protein mit Ähnlichkeit zu mtTFB, einem essentiellen Transkriptionsfaktor in
Hefemitochondrien, wurde MetA identifiziert. In in vitro-Assays initiierte RpoTm an
verschiedenen Promotoren die Transkription, während RpoTmp keine signifikante
Promotorspezifität zeigte. Die spezifische Promotornutzung durch RpoTm erforderte
superhelikale DNA. Weder RpoTm noch RpoTmp wurde durch MetA stimuliert. Eine mtTFB-
ähnliche Funktion von MetA ist daher unwahrscheinlich. Für MetA wurde ausserdem eine
engere phylogenetische Beziehung zu nukleären rRNA-Dimethylasen als zu mtTFB ermittelt.
Die hier vorgestellten Studien belegen die Transkription mitochondrialer Gene in Arabidopsis
durch RpoTm; für RpoTmp ist eine nicht-redundante Transkriptionsfunktion denkbar. Die
Kofaktor-unabhängige Spezifität von RpoTm für verschiedene Promotoren und die wenig
stringente Initiationskontrolle in vivo legen nahe, dass eine individuelle Regulation
mitochondrialer Gene in Arabidopsis auf Transkriptionsebene nicht erfolgt.

Schlagworte:
Pflanzenmitochondrien
Mitochondriengenom
Promotor
Transkription
Phagentyp-RNA-Polymerasen

Abstract
Mitochondria depend on a nucleus-encoded transcription machinery to express their genome.
The present study examined the transcription of mitochondrial genes by two nucleus-encoded
phage-type RNA polymerases, RpoTm and RpoTmp, in the plant Arabidopsis.
For selected mitochondrial genes in Arabidopsis, transcription initiation sites were
determined. Most genes were found to possess multiple promoters. The identified promoters
displayed diverse sequence elements and mostly deviated from a nonanucleotide consensus
derived previously for dicot mitochondrial promoters. Several promoters were detected that
activate transcription of presumably non-functional sequences. Promoter architecture,
distribution and utilization suggest a non-stringent control of transcription initiation in
Arabidopsis mitochondria.
An in vitro transcription system was set up to elucidate the roles of RpoTm and RpoTmp.
Since RpoT enzymes possibly require auxiliary factors, the Arabidopsis genome was screened
for potential cofactors of phage-type RNA polymerases. A mitochondrial protein (MetA) with
similarity to mtTFB, an essential transcription factor in yeast mitochondria, was identified. In in
vitro transcription studies, RpoTm recognized various promoters whereas RpoTmp displayed no
significant promoter specificity. Promoter recognition by RpoTm depended on supercoiled DNA
templates. Transcription initiation by RpoTm or RpoTmp was not affected by MetA, indicating
that MetA is not functionally equivalent to mtTFB. Besides, MetA was found to be more closely
related to non-mitochondrial rRNA dimethylases than to mtTFB.
The present study establishes RpoTm to transcribe mitochondrial genes; RpoTmp may have a
non-overlapping transcriptional role in mitochondria. The cofactor-independent promoter
specificity of RpoTm and the apparently non-stringent control of transcription initiation in vivo
imply that mitochondrial genes in Arabidopsis may not be regulated individually at the
transcriptional level.

Keywords:
Plant mitochondria
Mitochondrial genome
Promoter
Transcription
Phage-type RNA polymerase 1

Table of Contents
ZUSAMMENFASSUNG 4
SUMMARY 5
I INTRODUCTION 6
I.1 The mitochondrion, an endosymbiont-derived cell organelle 6
I.2 Plant mitochondrial genomes 7
I.3 Transcription in higher plant mitochondria 10
I.3.1 Mitochondrial promoters 10
I.3.2 Mitochondrial T7 bacteriophage-like RNA polymerases 12
I.3.2.1 Plant RpoT genes encoding phage-type transcriptases 12
I.3.2.2 Roles of RpoT enzymes 14
I.3.2.3 Structure of bacteriophage and phage-type RNA polymerases 15
I.3.3 Mitochondrial transcription factors 17
I.3.3.1 Yeast and animal mtTFB 18
I.3.3.2 al mtTFA 20
I.3.3.3 Mitochondrial transcription factors in plants 21
I.3.3.4 Cofactors of phage-type RNA polymerases in plastids 22
I.3.4 Regulation of mitochondrial gene expression at the transcriptional level 23
I.4 Aims of this study 25
II MATERIALS AND METHODS 26
II.1 Growth of Arabidopsis thaliana 26
II.2 Strains and culturing of Escherichia coli
II.3 Nucleic acids 26
II.3.1 Isolation of nucleic acids 26
II.3.1.1 Isolation of genomic DNA from Arabidopsis 26
II.3.1.2 Plasmid isolation from E. coli
II.3.1.3 Isolation of total RNA and mRNA-enriched RNA from Arabidopsis 26
II.3.2 Determination of nucleic acid concentrations 26
II.3.3 Nucleid acid electrophoreses 26
II.3.3.1 Agarose gel electrophoresis of DNA 26
II.3.3.2 Agarose gel electrophoresis of RNA 27
II.3.3.3 Denaturing polyacrylamide gel eletrophoresis (PAGE) of RNA 27
II.3.3.4 Native PAGE of DNA 27
II.3.4 cDNA synthesis and RT-PCR 28
II.3.5 PCR 28
II.3.6 Cloning and sequencing
II.3.6.1 Transformation of E. coli 28
II.3.6.2 Sequencing 28
II.3.7 5’-RACE analysis of RNA 29
II.3.8 Analysis of in vitro-cappable transcripts 32
II.3.8.1 Preparation of riboprobes 32
II.3.8.2 In vitro capping and RNase protection
II.4 Protein analysis 32
II.4.1 Determination of protein concentrations 32
II.4.2 SDS polyacrylamide gel electrophoresis (SDS PAGE) 33
II.4.3 Immunoblotting 33 2

II.5 Recombinant protein expression 34
II.5.1 Plasmids for the expression of recombinant proteins 34
II.5.2 Protein expression in E. coli 34
II.5.3 Purification of recombinant proteins from E. coli 35
II.5.3.1 Trx-(His) -tagged RpoTm and RpoTmp 35 6
II.5.3.2 Proteolytic removal of thioredoxin 36
II.5.3.3 (His) -tagged MetA and MetB 36 6
II.6 Electrophoretic mobility shift assay 36
II.6.1 Gel mobility shift probes 36
II.6.2 DNA binding assay 36
II.7 In vitro transcription 37
II.7.1 Template construction 37
II.7.2 In vitro transcription assay
II.7.3 5’-end mapping of in vitro-synthesized RNAs 38
II.8 Green fluorescent protein (GFP) import assay 38
II.8.1 GFP targeting constructs 38
II.8.2 Transient expression in tobacco protoplasts and microscopy 38
II.9 Alignments and phylogeny 39
II.10 Material 40
II.11 Providers 40
III RESULTS 41
III.1 Analysis of mitochondrial promoters in Arabidopsis thaliana 41
III.1.1 Identification of transcription initiation sites by 5’-RACE 41
III.1.2 Identification of transcription initiation sites by in vitro capping 46
III.1.3 Mitochondrial promoter architecture in Arabidopsis 49
III.1.4 Promoters directing transcription of non-coding sequences 50
III.2 Characterization of a mitochondrial mtTFB-like protein in Arabidopsis 54
III.2.1 Identification of mtTFB-like sequences in the Arabidopsis genome 54
III.2.2 Mitochondrial localization of the mtTFB-like protein MetA 56
III.2.3 Phylogenetic analysis of plant, fungal and animal rRNA dimethylase-like
proteins 57
III.2.4 Non-specific DNA binding by recombinant MetA 59
III.3 Expression of the Arabidopsis phage-type RNA polymerases RpoTm and
RpoTmp in E. coli 62
III.4 In vitro transcription studies of Arabidopsis RpoTm and RpoTmp 66
III.4.1 Development of an Arabidopsis in vitro transcription system 66
III.4.2 In vitro transcription from the mitochondrial promoters Patp6-1-200, PtrnM-98
and Prrn26-893 by RpoTm 67
III.4.3 Comparison of the transcriptional