Analysis of components of the mitochondrial transcription machinery in Arabidopsis thaliana

Analysis of components of the mitochondrial transcription machinery in Arabidopsis thaliana Kristina KühnII.11599#393I.22449#3963I.3I.3.14397#105I.3.2I.3.2.156435#2857I.3.2.28I.3.2.39568#497I.3.310I.3.3.1417#25811I.3.3.212I.3.3.313I.3.3.414I.3.415I.416IIII.117II.2II.3II.3.1II.3.1.1II.3.1.218II.3.1.3II.3.219II.3.3II.3.3.120II.3.3.2II.3.3.321II.3.3.422II.3.4II.3.523II.3.624II.3.6.1II.3.6.225II.3.726II.3.8II.3.8.127II.3.8.2II.4II.4.1II.4.228II.4.32930II.5II.5.131II.5.23233II.5.3II.5.3.1II.5.3.234II.5.3.3II.6II.6.135II.6.2II.7II.7.136II.7.2II.7.337II.8II.8.1II.8.238II.9II.1039II.11IIIIII.1III.1.13940306#246377#42441604#21042562#23443380#27844III.1.245381#693III.1.346295#189III.1.44748604#580411#289III.2III.2.14950III.2.251604#304III.2.352604#662III.2.453148#22254596#356Analysis of components of the mitochondrial transcription machinery in Arabidopsis thaliana DISSERTATIONzur Erlangung des akademischen Grades
doctor rerum naturalium (Dr. rer. nat.) im Fach Biologieeingereicht an der
Mathematisch-Naturwissenschaftlichen Fakultät Ider Humboldt-Universität zu Berlinvon
Diplom-Biologin Kristina Kühn
geboren am 27.08.1974 in Stollberg

Prof. Dr. Hans Jürgen Prömel
Präsident der Humboldt-Universität zu Berlin in Vertretung

Prof. Thomas Buckhout, PhD
Dekan der Mathematisch-Naturwissenschaftlichen Fakultät I Prof. Dr. Thomas Börner Prof. Dr. Wolfgang Hess 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-Transkriptionssystem 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.

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.

Plant mitochondria Mitochondrial genome Promoter Transcription Phage-type RNA polymerase Zusammenfassung

Im Zellkern kodierte RNA-Polymerasen sind für die Transkription der mitochondrialen Genome eukaryotischer Zellen verantwortlich und übernehmen somit eine zentrale Rolle in der mitochondrialen Genexpression. Für verschiedenen Pflanzenspezies wurden nukleäre T7-phagenähnliche RNA-Polymerasegene (RpoT-Gene) beschrieben, welche vermutlich für katalytische Untereinheiten der mitochondrialen Transkriptionsmaschinerie kodieren. Eine mitochondriale Transkriptionsfunktion von RpoT-Genprodukten in photosynthetisierenden Eukaryoten wurde jedoch bislang nicht nachgewiesen. In der vorliegenden Arbeit wurde die Transkription mitochondrialer Gene durch die mitochondrialen Phagentyp-RNA-Polymerasen RpoTm und RpoTmp der Pflanze Arabidopsis thaliana untersucht.

Im mitochondrialen Genom von Arabidopsis wurden Transkriptionsstartpunkte ausgewählter Gene und Gencluster bestimmt. Erstmals wurde hier für eine dikotyle Pflanze gezeigt, dass mitochondriale Gene häufig multiple Promotoren besitzen. Die identifizierten Promotoren zeigten verschiedene Sequenzelemente und wichen zumeist signifikant von der für Dikotyle publizierten Konsensussequenz ab. Es wurde darüber hinaus die Funktion von Promotoren nachgewiesen, welche die Transkription nichtkodierender und vermutlich nicht funktioneller Sequenzen aktivieren. Zwischen Promotoraktivitäten in Blüten- und in Blattgewebe wurden keine qualitativen Unterschiede beobachtet. Architektur, Häufigkeit, Lokalisation und Nutzung mitochondrialer Promotoren implizieren eine wenig stringente Kontrolle der Transkriptionsinitiation in Arabidopsis-Mitochondrien.

Die Identifizierung mitochondrialer Promotoren in Arabidopsis ermöglichte die Rekonstitution eines definierten in vitro-Transkriptionssystems zur Analyse der Transkription mitochondrialer Gene durch RpoTm und RpoTmp. In in vitro-Assays mit rekombinanten RNA-Polymerasen initiierte RpoTm an verschiedenen, jedoch nicht allen angebotenen Promotorsequenzen die Transkription, während für RpoTmp keine signifikante Promotorspezifität beobachtet wurde. Offenbar wird die Spezifität des mitochondrialen Transkriptionsapparates für zahlreiche Promotoren durch RpoTm vermittelt. RpoTm initiierte die in vitro-Transkription an Promotoren auf supercoiled-strukturierten, jedoch nicht auf linearen DNA-Molekülen und unterscheidet sich hierin von in früheren Arbeiten charakterisierten transkriptionsaktiven Extrakten aus Pflanzenmitochondrien. Dieser Befund impliziert, dass Kofaktoren, deren Funktion bei einer supercoiled-Konformation der DNA nicht essentiell ist, in in vitro-Assays mit mitochondrialen Extrakten und in vivo die Aufschmelzung der DNA unterstützen. Im Genom von Arabidopsis wurde nach Genen potentieller Kofaktoren von Phagentyp-RNA-Polymerasen gesucht. Ein durch den Kernlocus At5g66360 (MetA) kodiertes Protein, dessen mitochondriale Lokalisation hier nachgewiesen wurde, zeigt Ähnlichkeit zu mtTFB, einem essentiellem Kofaktor der T7-ähnlichen RNA-Polymerasen in Hefe- und Säugermitochondrien. In in vitro-Assays wurde die Initiation der RpoTm- und RpoTmp-abhängigen Transkription in keiner Weise durch MetA beeinflusst. Eine mtTFB-ähnliche Funktion von MetA in der mitochondrialen RNA-Synthese ist daher unwahrscheinlich. In Übereinstimmung hiermit wurde für MetA eine engere phylogenetische Beziehung zu nicht-mitochondrialen rRNA-Dimethylasen als zu mtTFB-Proteinen ermittelt.

Die hier vorgestellten Studien belegen die Transkription mitochondrialer Gene in Arabidopsis durch das Enzym RpoTm; für RpoTmp ist eine nicht-redundante mitochondriale Transkriptionsfunktion denkbar, welche die Erkennung bekannter Promotoren nicht erfordert. Im Pflanzenreich wird hiermit erstmals der funktionelle Nachweis erbracht, dass ein nukleäres Phagentyp-RNA-Polymerasegen für ein Transkriptionsenzym mit mitochondrialer Promotorspezifität kodiert. Die Kofaktor-unabhängige in vitro-Spezifität von RpoTm für verschiedene Promotorsequenzen und die offenbar nicht stringente Initiationskontrolle in vivo legen nahe, dass eine individuelle Regulation mitochondrialer Gene in Arabidopsis auf Transkriptionsebene nicht erfolgt.

Summary

Mitochondria depend on a nucleus-encoded transcription machinery to express their genome and maintain mitochondrial function. In plants, nuclear T7 bacteriophage-like RpoT genes identified in a variety of species have been suggested to encode catalytic subunits of the mitochondrial transcription apparatus. Still, functional evidence has been lacking for RpoT enzymes being involved in mitochondrial transcription in photosynthetic eukaryotes. The present study examined the transcription of mitochondrial genes by two mitochondrial phage-type RNA polymerases encoded by the RpoTm and RpoTmp genes in the plant Arabidopsis thaliana.

To study cis-elements that are recognized by these enzymes, transcription initiation sites of selected mitochondrial genes and gene clusters in Arabidopsis were determined. Most genes were found to possess multiple promoters, revealing for the first time that promoter multiplicity is a common feature of mitochondrial genes in a dicotyledonous plant. The identified promoters displayed diverse sequence elements and for the most part deviated significantly from a nonanucleotide consensus derived previously for dicot mitochondrial promoters. Several promoters were moreover detected that activate transcription of non-coding and presumably non-functional sequences. No qualitative differences in promoter utilization were observed between leaves and flowers. Promoter architecture, distribution and utilization suggest a non-stringent control of transcription initiation in Arabidopsis mitochondria.

The knowledge of mitochondrial promoters in Arabidopsis allowed a defined in vitro transcription system to be set up in order to elucidate the roles of RpoTm and RpoTmp in mitochondrial transcription. Since RpoT-driven transcription from mitochondrial promoters possibly requires a complementation of RpoT enzymes with as yet unidentified auxiliary factors, the Arabidopsis genome was screened for potential cofactors of phage-type RNA polymerases. A protein encoded by the nuclear locus At5g66360 (MetA) and shown here to be imported into mitochondria displays sequence similarity to mtTFB, an essential cofactor of the mitochondrial T7-like RNA polymerase in yeast and mammals that is related to rRNA methyltransferases. In vitro transcription studies examined the abilities of recombinant RpoTm and RpoTmp to transcribe DNA from mitochondrial promoters, and moreover tested MetA for its potential to modulate the transcriptional performances of RpoTm and RpoTmp. RpoTm recognized a variety of promoters whereas RpoTmp displayed no significant promoter specificity. Sequence specificity of the mitochondrial transcription apparatus thus appears to be conferred by the RpoTm core enzyme for the majority of promoters. RpoTm differed in its transcriptional performance from formerly characterized plant mitochondrial extracts in that it did not specifically initiate transcription at promoters located on linear DNA but required supercoiled templates. This indicates a participation of (an) auxiliary factor(s) in DNA melting in transcription experiments using mitochondrial extracts and in vivo, which is obviated by a supercoiled DNA conformation. Transcription initiation by RpoTm or RpoTmp appeared to be not affected by the presence of MetA in the in vitro assay, indicating that MetA does not have a role equivalent to that of yeast or mammalian mtTFB in mitochondrial RNA synthesis. In line with this, phylogenetic analyses revealed MetA to be more closely related to a group of non-mitochondrial rRNA dimethylases than to fungal or animal mtTFBs.

The data presented here establish RpoTm to transcribe mitochondrial genes. They thus provide the first direct linkage in the plant kingdom between an RNA polymerase activity recognizing mitochondrial promoters and a nuclear gene encoding a mitochondrial phage-type RNA polymerase. Experimental results suggest RpoTmp to have a non-overlapping transcriptional role in mitochondria, which might not involve the recognition of known mitochondrial promoters. The cofactor-independent in vitro specificity of RpoTm for diverse promoter sequences 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.

Table of contents

I Introduction
- I.1 The mitochondrion, an endosymbiont-derived cell organelle
- I.2 Plant mitochondrial genomes
- I.3 Transcription in higher plant mitochondria
  - I.3.1 Mitochondrial promoters
  - I.3.2 Mitochondrial T7 bacteriophage-like RNA polymerases
    - I.3.2.1 Plant RpoT genes encoding phage-type transcriptases
    - I.3.2.2 Roles of RpoT enzymes
    - I.3.2.3 Structure of bacteriophage and phage-type RNA polymerases
  - I.3.3 Mitochondrial transcription factors
    - I.3.3.1 Yeast and animal mtTFB
    - I.3.3.2 Yeast and animal mtTFA
    - I.3.3.3 Mitochondrial transcription factors in plants
    - I.3.3.4 Cofactors of phage-type RNA polymerases in plastids
  - I.3.4 Regulation of mitochondrial gene expression at the transcriptional level
- I.4 Aims of this study
II Materials and Methods
- II.1 Growth of Arabidopsis thaliana
- II.2 Strains and culturing of Escherichia coli
- II.3 Nucleic acids
  - II.3.1 Isolation of nucleic acids
    - II.3.1.1 Isolation of genomic DNA from Arabidopsis
    - II.3.1.2 Plasmid isolation from E. coli
    - II.3.1.3 Isolation of total RNA and mRNA-enriched RNA from Arabidopsis
  - II.3.2 Determination of nucleic acid concentrations
  - II.3.3 Nucleid acid electrophoreses
    - II.3.3.1 Agarose gel electrophoresis of DNA
    - II.3.3.2 Agarose gel electrophoresis of RNA
    - II.3.3.3 Denaturing polyacrylamide gel eletrophoresis (PAGE) of RNA
    - II.3.3.4 Native PAGE of DNA
  - II.3.4 cDNA synthesis and RT-PCR
  - II.3.5 PCR
  - II.3.6 Cloning and sequencing
    - II.3.6.1 Transformation of E. coli
    - II.3.6.2 Sequencing
  - II.3.7 5’-RACE analysis of RNA
  - II.3.8 Analysis of in vitro-cappable transcripts
    - II.3.8.1 Preparation of riboprobes
    - II.3.8.2 In vitro capping and RNase protection
- II.4 Protein analysis
  - II.4.1 Determination of protein concentrations
  - II.4.2 SDS polyacrylamide gel electrophoresis (SDS PAGE)
  - II.4.3 Immunoblotting
- II.5 Recombinant protein expression
  - II.5.1 Plasmids for the expression of recombinant proteins
  - II.5.2 Protein expression in E. coli
  - II.5.3 Purification of recombinant proteins from E. coli
    - II.5.3.1 Trx-(His)₆-tagged RpoTm and RpoTmp
    - II.5.3.2 Proteolytic removal of thioredoxin
    - II.5.3.3 (His)₆-tagged MetA and MetB
- II.6 Electrophoretic mobility shift assay
  - II.6.1 Gel mobility shift probes
  - II.6.2 DNA binding assay
- II.7 In vitro transcription
  - II.7.1 Template construction
  - II.7.2 In vitro transcription assay
  - II.7.3 5’-end mapping of in vitro-synthesized RNAs
- II.8 Green fluorescent protein (GFP) import assay
  - II.8.1 GFP targeting constructs
  - II.8.2 Transient expression in tobacco protoplasts and microscopy
- II.9 Alignments and phylogeny
- II.10 Material
- II.11 Providers
III Results
- III.1 Analysis of mitochondrial promoters in Arabidopsis thaliana
  - III.1.1 Identification of transcription initiation sites by 5’-RACE
  - III.1.2 Identification of transcription initiation sites by in vitro capping
  - III.1.3 Mitochondrial promoter architecture in Arabidopsis
  - III.1.4 Promoters directing transcription of non-coding sequences
- III.2 Characterization of a mitochondrial mtTFB-like protein in Arabidopsis
  - III.2.1 Identification of mtTFB-like sequences in the Arabidopsis genome
  - III.2.2 Mitochondrial localization of the mtTFB-like protein MetA
  - III.2.3 Phylogenetic analysis of plant, fungal and animal rRNA dimethylase-like proteins
  - III.2.4 Non-specific DNA binding by recombinant MetA
- III.3 Expression of the Arabidopsis phage-type RNA polymerases RpoTm and RpoTmp in E. coli
- III.4 In vitro transcription studies of Arabidopsis RpoTm and RpoTmp
  - III.4.1 Development of an Arabidopsis in vitro transcription system
  - III.4.2 In vitro transcription from the mitochondrial promoters Patp6-1-200, PtrnM-98 and Prrn26-893 by RpoTm
  - III.4.3 Comparison of the transcriptional performances of RpoTm and RpoTmp
  - III.4.4 Transcription initiation by RpoTm and RpoTmp at non-CRTA promoters
IV Discussion
- IV.1 Multiple promoters as a common feature of mitochondrial genes in Arabidopsis
  - IV.1.1 Identification of transcription initiation sites
  - IV.1.2 Promoter architecture
  - IV.1.3 Promoter distribution
  - IV.1.4 Non-stringent control of Arabidopsis mtDNA transcription
- IV.2 Potential mtTFB-like cofactors of phage-type RNA polymerases in Arabidopsis
  - IV.2.1 A mtTFB-like protein in Arabidopsis mitochondria
  - IV.2.2 Phylogenetic relationship between mtTFB and related rRNA dimethylases
  - IV.2.3 Implications of the function of homologous RNA polymerases in mitochondria and plastids
- IV.3 Transcriptional roles of the phage-type RNA polymerases RpoTm and RpoTmp in Arabidopsis mitochondria
  - IV.3.1 Development of an Arabidopsis in vitro transcription system
  - IV.3.2 Intrinsic promoter specificity of RpoTm
  - IV.3.3 Different transcriptional properties of RpoTm and RpoTmp
- IV.4 Does a mtTFB homologue function in mitochondrial transcription in plants?
- IV.5 Transcription initiation in Arabidopsis mitochondria
References
Abbreviations
Annex
Curriculum Vitae
Publications and Conference Abstracts
Danksagung
Eidesstattliche Erklärung

Tables

Table 1: Oligonucleotides used for sequencing.
Table 2: mtDNA-specific primers used for 5’-RACE.
Table 3: Primers used for cRNA synthesis template construction.
Table 4: Antisera.
Table 5: Oligonucleotides used to construct plasmids driving recombinant protein expression.
Table 6: Construction of in vitro transcription templates.
Table 7: Primers used for 5’-end mapping of in vitro-synthesized RNAs
Table 8: Oligonucleotides used for amplification of MetA and MetB N-termini.
Table 9: Transcription initiation sites detected by 5’-RACE and in vitro-capping.
Table 10: Transcription initiation sites associated with antisense and non-coding transcripts.
Table 11: Predicted properties of Arabidopsis MetA and MetB.
Table 12: Promoter recognition by RpoTm and RpoTmp in vitro.

Images

Figure 1: Participation of mtDNA-encoded proteins and RNAs in biological processes in mitochondria.
Figure 2: Distribution of identified genes in the mitochondrial genome of Arabidopsis. (Taken from (Dombrowski, et al., 1998).
Figure 3: Conserved sequence elements of plant mitochondrial promoters.
Figure 4: Nuclear genes encoding organellar phage-type RNA polymerases.
Figure 5: Conserved sequence regions of the T7 phage and eukaryotic phage-type RNA polymerases.
Figure 6: Components of the transcription machineries in yeast and human mitochondria.
Figure 7: 5’-RACE technique used to distinguish primary from processed transcript 5’ termini.
Figure 8: 5’-RACE analysis of the mitochondrial rrn18, cox2 and atp9 transcripts.
Figure 9: 5’ end identification by sequencing across ligation sites of 5’-RACE products.
Figure 10: Transcript 5’ termini detected through cloning and sequencing of 5’-RACE products designated Pcox2-210, Pcox2-481, Patp9-239 and Patp9-295 in Figure 8.
Figure 11: 5’-RACE analysis of the mitochondrial rrn18, rps3 and atp6-1 transcripts.
Figure 12: Detection of selected rrn18, rrn26, atp6-1 and atp9 primary transcript 5’ ends by ribonuclease protection of cap-labelled RNA.
Figure 13: Summary of nucleotide sequences around experimentally defined transcription initiation sites in Arabidopsis mitochondria, as displayed in Table 9.
Figure 14: Synthesis of antisense transcripts to mitochondrial nad genes.
Figure 15: Synthesis of transcripts from intergenic regions of the Arabidopsis mtDNA.
Figure 16: Transient expression of GFP fusion proteins in tobacco protoplasts.
Figure 17: Phylogeny of mitochondrial transcription factors and small-subunit rRNA dimethylases.
Figure 18: Purification of recombinant MetA and MetB.
Figure 19: Gel mobility shift competition assay showing non-specific DNA binding by MetA and MetB.
Figure 20: Purification of recombinant RpoTm and RpoTmp.
Figure 21: Proteolytic removal of the Trx domain from Trx-(His)₆-tagged RpoTm and RpoTmp.
Figure 22: Expression of untagged RpoTm and RpoTmp in E. coli.
Figure 23: In vitro run-off transcription of pKL23-atp6-1-A by RpoTm initiates at Patp6-1-200.
Figure 24: 5’-RACE analysis of pKL23-atp6-1-A-derived transcripts synthesized in vitro by RpoTm.
Figure 25: In vitro transcription from PtrnM-98 and Prrn26-893 by RpoTm.
Figure 26: 5’-RACE analysis of RNAs transcribed from PtrnM-98 and Prrn26-893 by RpoTm in vitro.
Figure 27: Maps of in vitro transcription templates.
Figure 28: Run-off transcription from the atp6-1, atp8, rrn18 and cox2 upstream regions by RpoTm and RpoTmp.
Figure 29: 5’-RACE analysis of in vitro-synthesized transcripts indicated in Figure 28.
Figure 30: In vitro transcription initiation at promoters not displaying a CRTA sequence element.
Figure 31: Linear diagram of upstream regions of the mitochondrial genes trnM, rrn18, rrn26, rps3, cox1, cox2, atp1, atp6-1, atp6-2, atp8, atp9 and orf291.
Figure 32: Comparison of the specificity loop region of phage and phage-type RNA polymerases.
Figure 33: Transcription initiation in Arabidopsis mitochondria.