©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
A Novel Pea Mitochondrial in Vitro Transcription System Recognizes Homologous and Heterologous mRNA and tRNA Promoters (*)

(Received for publication, April 24, 1995; and in revised form, June 21, 1995)

Stefan Binder (1)(§) Frank Hatzack (1) Axel Brennicke (2)

From the  (1)Institut für Genbiologische Forschung, Ihnestrasse 63, D-14195 Berlin and the (2)Institut für Allgemeine Botanik, Universität Ulm, Albert-Einstein-Allee 11, D-89069 Ulm, Federal Republic of Germany

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

To elucidate the mechanism involved in the transcription initiation process in mitochondria of dicotyledonous plants, an in vitro transcription system was established for pea (Pisum sativum L.). The partially purified mitochondrial protein extract initiates transcription on homologous pea templates as well as on heterologous mitochondrial DNA from other dicot plant species. In vitro transcription begins within the nonanucleotide 5`-CRTAAGAGA-3` (transcription start site is underlined) conserved at most of the identified transcription initiation sites in dicot plant mitochondria. The in vitro initiation at promoters of protein as well as of tRNA coding genes indicates a common mode of transcription initiation for different types of RNA. The competent recognition of different heterologous templates supports a general functional role of the conserved nonanucleotide within mitochondrial promoters of dicotyledonous plants. Initial studies of the promoter structure by deletion analysis in the 5` region of the pea atp9 promoter show that in addition to the conserved nonanucleotide, which is essential for transcription initiation in vitro, sequences up to 25 nucleotides upstream of the transcription start site are necessary for an efficient initiation event.


INTRODUCTION

The resident mitochondrial genomes encode essential genes, whose expression is indispensable for the function of the mitochondria and thus for the survival of the eukaryotic cell (Levings and Brown, 1989). Towards understanding the regulatory control mechanisms involved in mitochondrial gene expression, the biochemistry of transcription of the mitochondrial DNA has been intensively studied in animals and fungi. The mammalian 16-kilobase mitochondrial genome is symmetrically transcribed from two major promoters, one for each of the two different DNA strands. Transcription of the Saccharomyces cerevisiae mitochondrial DNA is initiated at about 20 copies of a highly conserved promoter motif (Christianson and Rabinowitz, 1983). In vitro transcription studies showed that in both yeast and mammalia at least two protein components are engaged in specific and efficient transcription initiation. Although in yeast all genes and the encoded proteins of the mitochondrial transcription machinery are described in detail, only the gene for the transcription factor h-mtTFA has been identified in human mitochondria (Shadel and Clayton, 1993).

Compared with animals and fungi the mode of transcription initiation in mitochondria of plants remains elusive. Their much larger and more complex mitochondrial genomes and the frequently observed complex transcription patterns of individual genes impede the identification and analysis of promoters in mitochondria of plants (Levings and Brown, 1989; Newton, 1988). Several transcription initiation sites have clearly been identified by in vitro capping analyses in mitochondria from both monocot and dicot plant species. Inspection of sequences at these potential promoter regions identified distinct sequence motifs to which promoter function has been attributed (Mulligan et al., 1988a, 1988b, 1991; Covello and Gray, 1991; Brown et al., 1991; Binder and Brennicke, 1993a, 1993b).

The successful development of in vitro transcription systems for the monocot plant species wheat and maize confirms the competence of several promoters of protein coding genes in vitro (Hanic-Joyce and Gray, 1991; Rapp and Stern, 1992). Detailed investigation by deletion analysis, linker-scanning mutagenesis, and site-directed mutagenesis revealed the atp1 promoter in maize to be composed of a central domain extending from -7 to +5 and a 3-base pair upstream domain located between positions -10 and -12. The central element contains the highly conserved 5`-YRTA-3` core element that is consistent with the 5`-CRTA-3` motif defined by sequence inspection at the transcription initiation sites (Rapp et al., 1993; Mulligan et al., 1991). This motif is observed at most of the identified monocot mitochondrial promoters, which otherwise show only limited similarity between different species (Mulligan et al., 1991; Gray et al., 1992).

In dicot plant mitochondria, in vitro capping analysis of primary transcripts indicates a much better conservation of most of the potential promoter sequences. A conserved nonanucleotide motif 5`-CRTAAGAGA-3` has been defined by comparison of sequences surrounding mRMA, rRNA, and tRNA transcription initiation sites. With monocot promoters, only the tetranucleotide 5`-CRTA-3` appears to be conserved. Because the nonanucleotide motif is common to transcription initiation sites of different dicot plant species, it may represent a general core element of mitochondrial promoters in this plant group (Brown et al., 1991; Binder and Brennicke, 1993a, 1993b). However, several transcription initiation sites have been determined that show no sequence similarity to this conserved nonanucleotide motif or any other known plant mitochondrial promoter. Structure and function of these most likely gene- and/or species-specific potential promoters remain unclear (Brown et al., 1991; Binder et al., 1994).

To extend our knowledge about the mode of transcription initiation in dicot plant mitochondria we have now established an in vitro transcription system for pea. The in vitro results indicate the conserved nonanucleotide motif to be essential for transcription initiation and confirm its function as a general promoter motif in different dicot plant species. In addition to mRNA promoters the pea mitochondrial extract initiates transcription at a tRNA promoter suggesting that the transcription activity in this in vitro system is not restricted to protein coding genes.


MATERIALS AND METHODS

Recombinant DNA Templates

Plasmid clones patp9SC550 and patp9SK630 contain sequences covering the most distal 5` mRNA end of the atp9 gene subcloned from the type I allele of the 4 different atp1/atp9 arrangements present in pea mitochondrial DNA (Morikami and Nakamura, 1987, 1993). For deletion analysis of the pea atp9 promoter region, DNA fragments were amplified by PCR (^1)using patp9SK630 as template and the following oligonucleotides: clone Deltapatp9-67, dp-67 (5`-GTGGATCCTTATGTGAGGTTCTTTCC-3`) and dp+355 (5`-TGGGTACCTCATAGGGC-3`); clone Deltapatp9-35, dp-35 (5`-GTGGATCCTTGTTTTGAGTACTCGAC-3`) and dp+355; clone Deltapatp9-25, dp-25 (5`-GTGGATCCTACTCGACGAAATAATAG-3`) and dp+355; clone Deltapatp9-7, dp-7 (5`-GTGGATCCCATAATAAGAGAAGATATTGG-3`) and dp+355; and clone Deltapatp9+2, dp+2 (5`-GTGGATCCAGATATTGGACAATTGAG-3`) and dp+355. Designation of the upstream oligonucleotides reflects, with respect to the transcription start site, the position of the most 5` nucleotide within the oligonucleotide, which is identical with the mitochondrial sequence. A BamHI restriction site was introduced into each upstream oligonucleotide for cloning. The downstream oligonucleotide dp+355 covers a KpnI site contained in the mitochondrial DNA sequence. Amplified DNA fragments were digested with the respective restriction enzymes and cloned into pBluescriptII KS(-) vectors.

The soybean mtDNA clones KM57BK3 and KM2F2EH containing transcription initiation sites for the atp9 and RNA b/c gene, respectively, were kindly provided by Dr. Gregory Brown (Brown et al., 1991). Clone satp9XR482 is a 0.48-kilobase XbaI/RsaI sublone of KM57BK3.

Clone opheBH500 contains the transcription initiation site identified upstream of the Oenothera tRNA gene on a DNA fragment obtained by PCR with oligonucleotides OP-1 (5`-GGAAATCCAAGGAGGTGGC-3`) and P7 (5`-ATAAGCTTGAATTTCCAAATCCGG-3`).

All mtDNA fragments were cloned into pBluescript (Stratagene) vectors using standard cloning techniques (Sambrook et al., 1989). For in vitro transcription reactions plasmid DNA was purified by centrifugation on CsCl gradients and linearized with restriction enzymes. After digestion, template DNA was checked on agarose gels for complete cleavage, extracted with phenol/chloroform, precipitated with ethanol, and resuspended in double distilled water. DNA concentrations were determined photometrically by absorption at 260 nm.

Preparation of Mitochondrial Protein Extracts

Pea seedlings (Pisum sativum L., var. Progress No. 9 and var. Lancet) were grown in the dark for 7 days. Mitochondria were isolated by differential centrifugation and purification on Percoll gradients as described previously (Binder and Brennicke, 1993a). The mitochondrial protein extract was prepared basically following a method established by Hanic-Joyce and Gray(1991) for wheat and modified by Rapp and Stern(1992) for maize mitochondria. About 5 g of purified mitochondria were lysed in the presence of 0.5% Triton X-100 and 1 M KCl. Membranes were pelleted by centrifugation at 100,000 times g. The supernatant (S100) was diluted with 1 volume of buffer V containing 90 mM Tris-HCl, pH 8.0, 0.1 mM EDTA, 0.5 mM dithiothreitol, and 2.5% glycerol, and hydrophobic proteins were precipitated by the addition of 0.224 g of solid (NH(4))(2)SO(4)/ml of diluted solution and centrifugation at 16,000 times g for 30 min (20% AS fraction). The remaining protein was precipitated from the supernatant by the addition of 0.41 g of solid (NH(4))(2)SO(4)/ml of solution and centrifugation at 16,000 times g for 90 min (50% AS fraction). The protein pellet was then resuspended in 1 ml of buffer A (10 mM Tris-HCl pH 8.0, 1 mM dithiothreitol, 0.1 mM EDTA, and 7.5% glycerol) containing 50 mM KCl and dialyzed for several hours against 3 liters of the same buffer. For further purification by anion-exchange chromatography, this protein fraction was applied to a MonoQ column (Pharmacia Biotech Inc.) equilibrated with buffer A (with 50 mM KCl added). Bound protein was eluted from the column with 100, 200, 250, 300, 400, 500, and 750 mM KCl in buffer A. Fractions of each elution step were pooled, dialyzed against 3 liters of buffer A containing 10 mM KCl, concentrated by ultrafiltration using Centricon 10 (Amicon), rapidly frozen in liquid nitrogen, and stored at -80 °C. Protein concentrations were measured using a Bio-Rad (Bradford) protein assay.

In Vitro Transcription Assays

The in vitro transcription reactions were performed in a total volume of 12.5 µl of reaction mixture containing 10 mM Tris-HCl, pH 7.9, 10 mM MgCl(2), 1 mM dithiothreitol, 20 mM KCl, 500 µM each of ATP, CTP, and GTP, 25 µM UTP, 40 units of RNase inhibitor (Boehringer Mannheim), 10 µCi of [alpha-P]UTP (3000 Ci/mmol), and 100-500 ng of linearized template DNA. Reactions were started by the addition of 30-80 µg of the protein extract and were incubated for 30 min at 30 °C. After the addition of 37 µl of stop mix (4.8 M urea, 0.4 M sodium acetate, 5.3 mM aurintricarboxylic acid, 30 µg/ml tRNA (wheat germ), and 0.8% (w/v) SDS), reaction mixes were extracted with phenol/chloroform, and nucleic acids were precipitated with ethanol. Total nucleic acids were resuspended in 4 µl of loading solution (80% (v/v) formamide, 50 mM Tris borate, pH 8.3, 1 mM EDTA, 0.1% (w/v) xylene cyanol, and 0.1% (w/v) bromphenol blue) and electrophoresed on 5% polyacrylamide gels, and transcription products were examined by autoradiography.

Primer Extension Analysis

Primer extension analysis was carried out with in vitro transcripts recovered from the polyacrylamide gels in a buffer containing 500 mM ammonium acetate, 0.1 mM EDTA, and 0.1% (w/v) SDS. RNA was resuspended in 7 µl of double distilled water and mixed with 5 µl of 5` end-labeled primer (5 times 10^5 cpm). The mixture was incubated for 10 min at 70 °C and for another 10 min at 42 °C. After the addition of 2 µl of 10 times synthesis buffer, 2 µl 4 mM dNTP mix and 2 µl of 0.1 mM dithiothreitol, the reaction was started with 1 µl of Superscript reverse transcriptase (200 units/µl, Life Technologies, Inc.) and incubated for 1 h at 42 °C. Reaction products were precipitated with ethanol, resuspended in 4 µl of loading solution, and analyzed by polyacrylamide gel electrophoresis. Primer extension analysis of the in vitro transcripts of pea atp9 template patp9SK630 was performed using oligonucleotide PA-14 (5`-CAAAGGAGGAACTCCCG-3`) complementary to sequences between nucleotides +49 and +65 (first transcribed nucleotide is +1). Oligonucleotides A9 (5`-GTGAACAGAAGCTTTCTCGG-3`) and SBC-1 (5`-CTGCACACCAAGCCAATCTCG-3`) complementary to sequences between nucleotides +72 and +91 (atp9) and nucleotides +71 and +91 (RNA b/c), respectively, were used for the primer extension analysis of the soybean atp9 and RNA b/c in vitro transcripts.


RESULTS

Development of an in Vitro Transcription System for Dicot Plant Mitochondria

The recent identification of several mitochondrial transcription initiation sites of soybean and Oenothera by in vitro capping analysis (Brown et al., 1991; Binder and Brennicke, 1993a, 1993b) provided the DNA templates required for the preparation of a transcriptionally active mitochondrial extract. The isolation of such transcriptionally active extracts from dicot plant mitochondria was additionally guided by the successful development of an in vitro transcription system for monocot plants (Hanic-Joyce and Gray, 1991; Rapp and Stern, 1992). Because data on functional transcription initiation sites were only available for Oenothera and soybean, the isolation of transcriptionally active protein fractions was started in these two plant species. It soon became evident, however, that the impossibility of isolating large quantities of mitochondria from Oenothera or soybean and the high content of nucleases in protein extracts of these plant species prohibited the large scale preparation of transcriptionally active lysates. Attempts to prepare protein extracts from large amounts of mitochondria isolated from potato tubers likewise did not yield the desired activities, possibly owing to the basically low transcriptional activity in resting tissues like tubers (data not shown).

The search for a suitable dicot plant species and tissue type was finally successful in identifying pea seedlings as a suitable source for reasonable quantities of transcriptionally active mitochondria. Because no in vitro capping analysis had been carried out with pea transcripts, only little data were available on transcription initiation sites in mitochondria of this plant species. Homologous transcription assays thus relied on a DNA fragment containing the most distal 5` mRNA terminus mapped in the upstream region of the atp9 gene (Fig. 1A, terminus (1) (Morikami and Nakamura, 1993). The sequence surrounding this 5` end (5`-CATAAGAGA-3`), which was determined by primer extension and S1 protection analysis, shows high similarity with the conserved nonanucleotide element detected at primary 5` transcript termini in mitochondria of other dicot plants (Brown et al., 1991; Binder and Brennicke, 1993a, 1993b).


Figure 1: Enrichment procedure for promoter specific in vitro transcription activity in pea mitochondrial extracts. A, structure of the genomic fragment containing the atp9 coding region present in the type I allele in pea mitochondria (Morikami and Nakamura, 1987, 1993). Three 5` ends ((1)-(3), indicated by bent arrows) have been mapped upstream of the coding region. A SfuI/KpnI fragment containing the most distal 5` end ((1)) located 1048 nt upstream of the start codon was subcloned (patp9SK630) and used for in vitro transcription reactions. A 355-nt-long run-off transcript (dotted arrow) is expected for correct transcription initiation on the KpnI-linearized DNA template. The sequence from -7 to +2 surrounding this 5` end (underlined) shows high similarity to the putative promoter consensus sequence from mitochondria of dicot plants (Brown et al., 1991; Binder and Brennicke, 1993a, 1993b). Restriction sites are indicated for ClaI (C), KpnI (K), and SfuI (Sf). B, gel analysis of in vitro transcription assays with different fractions of the pea mitochondrial protein extracts and KpnI-linearized patp9SK630 DNA templates. Fractions used in the assays are: lysed mitochondria (mitochondria), S100 supernatant (S100), 20% and 50% ammonium sulfate fractions (20% AS and 50% AS), and fractions eluted from the MonoQ column with 50, 100, 200, 250, 300, 400, 500 and 750 mM KCl. Sizes of DNA length standards are given in nucleotides.



Specific Transcription Initiation by a Pea Mitochondrial Protein Extract

The KpnI-linearized patp9SK630 template containing the region described above (Fig. 1A) was used in homologous in vitro transcription assays to test all protein fractions obtained during the enrichment procedure (see ``Materials and Methods''). A weak signal corresponding to a transcript of the predicted size (Fig. 1A, 355 nt) was detected in the assay performed with the 50% ammonium sulfate fraction (Fig. 1B, 50% AS). The additional fractionation of the 50% AS cut resulted in enrichment of the specific transcription initiation activity. The majority of the specific activity could be detected in the 250 mM KCl MonoQ fraction represented by the signal in the size range expected for the correct run-off transcript. Additional weaker transcription initiation activity was also present in the 300 mM KCl MonoQ fraction, as indicated by a faint band of the predicted size (Fig. 1B, 250 and 300 mM KCl).

No specific transcription products were observed in assays with only vector sequences or without any DNA template, indicating their dependence upon mitochondrial promoter sequences. The absence of any transcription product in a reaction carried out in the presence of RNase A showed the products of these assays to be indeed RNA resulting from genuine transcription events (data not shown). Because the majority of the specific transcription initiation activity eluted at 250 mM KCl, this fraction was used for the detailed investigation of the accuracy of the initiation process in vitro and for heterologous transcription assays.

To confirm that transcription is indeed initiated within the mitochondrial insert, two differently linearized pea atp9 templates (patp9SK630 and patp9SC550, see ``Material and Methods'') were tested in the transcription assays. In all instances run-off transcripts of the predicted sizes were observed in these reactions, and the lengths of the in vitro synthesized RNAs differed by the distances of the respective restriction sites used for linearization of the DNA templates confirming correct initiation within the mitochondrial insert (data not shown).

Transcription Is Initiated on Heterologous Templates from Soybean

The conservation of the potential promoter sequences identified by in vitro capping analysis of soybean and Oenothera transcripts and by the above in vitro studies of the pea atp9 gene indicates similar transcription initiation complexes most likely including homologous protein components within these dicot plant species. This hypothesis was tested by heterologous transcription assays with mitochondrial extracts and DNA templates from different dicot plant species. Clones KM57BK3 and satp9XR482 containing the mitochondrial promoter of the soybean atp9 gene were assayed for transcription initiation in pea protein extracts (Fig. 2A). Both templates direct transcription of RNAs corresponding to the predicted sizes, indicating accurate initiation in these heterologous reactions (Fig. 2B, lanes 1 and 2).


Figure 2: The soybean atp9 promoter is recognized by the pea mitochondrial in vitro transcription system. A, a transcription initiation site (marked by a bent arrow) has been mapped by in vitro capping analysis upstream of the soybean atp9 gene (Brown et al., 1991). The 0.5-kilobase XbaI/RsaI and 1.69-kilobase BamHI/KpnI fragments, both containing this promoter, were subcloned (satp9XR482 and KM57BK3) and used for in vitro transcription reactions with the 250 mM KCl fraction of the pea mitochondrial extracts. Sizes of the run-off products (dotted lines) expected from transcription of KpnI-linearized DNA templates are 280 and 238 nt, respectively. Vector sequences are indicated by dashed lines; restriction sites are given for BamHI (B), XbaI (X), KpnI (K), and RsaI (R). B, gel analysis of run-off transcripts transcribed from the DNA templates as shown above. Numbering of the gel lanes correlates with the expected run-off transcripts. Signals corresponding to run-off transcripts of the predicted sizes are observed in both reactions. DNA length standards are given in nucleotides.



In addition to the atp9 promoter clone, KM2F2EH carrying two transcription initiation sites for RNAs of unidentified function (RNA b, c, and e) (Brown et al., 1991) was subjected to heterologous assays (Fig. 3). Although the common transcription initiation site for RNA b and c conforms to the consensus sequence element, sequences at the transcription start site for RNA e show no similarity to this promoter motif (Brown et al., 1991). Transcripts with the sizes predicted for correct initiation at the promoter for RNA b and c were observed with two differently linearized templates (Fig. 3). However, no transcripts of the respective sizes were observed for RNA e, suggesting that this promoter is not recognized by the pea extract.


Figure 3: Specificity of the pea mitochondrial extract for promoters containing the conserved nonanucleotide motif. A, the genomic location containing two transcription start points (bent arrows) for three different RNAs in soybean mitochondria (Brown et al., 1991). The initiation sites are indicated by bent arrows and designated (b/c) and (e) corresponding to the transcribed RNAs b/c and e. Transcription initiation at start point (b/c) should yield run-off transcripts of 330 and 298 nt (dotted arrows) on an EcoRI/HindIII clone (KM2F2EH) linearized with KpnI (site located in the vector sequence) and HindIII, respectively. Restriction sites are indicated for EcoRI (E), HindIII (H), and KpnI (K). B, run-off transcripts checked by polyacrylamide gel electrophoresis show correct transcription initiation at site (b/c) in the in vitro transcription reactions. Lanes with the transcription assays are numbered corresponding to the expected run-off transcripts shown in A. Transcripts of the sizes expected from transcription initiation at site (e) are not observed in these assays. Sizes of coelectrophoresed DNA length standards are given in nucleotides.



To extend the analysis of heterologous transcription initiation, cox2 and atp1 templates from Oenothera were tested with the pea extract. In these assays distinct albeit weak signals corresponding to run-off transcripts of the predicted sizes showed correct recognition of these heterologous templates by the pea extract (data not shown).

In Vitro Transcription of a tRNA Gene Promoter

In vitro capping analysis of primary transcripts covering tRNA genes encoded in Oenothera mitochondria had identified a transcription initiation site located upstream of three alleles of the gene for tRNA and a gene for tRNA. The sequence at this transcription initiation site is consistent with the conserved sequence element derived from protein coding and rRNA genes (Binder and Brennicke, 1993b). To test whether this promoter is also recognized and transcribed by the pea extract, clone opheBK500 containing a PCR-amplified 500-nt-long DNA fragment with the complete gene for tRNA was used in the transcription analysis. A run-off transcript of 301 nt is expected from correct transcription initiation at this tRNA promoter and full-length elongation along the HindIII-cleaved template (Fig. 4A). Analysis of the transcription products by polyacrylamide gel electrophoresis shows a RNA of the expected size to be synthesized in the in vitro reaction (Fig. 4B). This result confirms common features for promoters of both protein coding and tRNA genes.


Figure 4: A promoter located upstream of a tRNA gene in Oenothera mitochondria is recognized in the pea in vitro transcription system. A, a cloned PCR product (opheBK500) containing a copy of this gene and about 380 nt of the upstream region was used for in vitro transcription assays with the pea extract. The correct recognition of this potential promoter (marked by a bent arrow) on a HindIII-cleaved template should result in the transcription of a 301-nt-long RNA (dotted arrow). A restriction site is indicated for HindIII (H). S/Bl marks the blunt end of the DNA fragment cloned in the SmaI restriction site of the vector (dashed lines). B, gel analysis of in vitro transcription experiments shows a run-off transcript of the predicted size. Sizes of coelectrophoresed DNA length standards are given in nucleotides.



Precise Mapping of 5` Ends of in Vitro Synthesized RNAs

The accuracy of the in vitro transcription initiation event was determined by primer extension analysis carried out with specific transcripts synthesized in both homologous and heterologous in vitro transcription reactions.

In the analysis of homologous pea atp9 transcripts, the 5` ends of the in vitro transcribed RNAs are scattered around the transcription start site used in vivo. In addition to the 5` end of the correctly initiated transcript corresponding to the primer extension length obtained on in vivo transcripts (data not shown), signals corresponding to RNAs up to eight nucleotides longer were detected in this analysis (Fig. 5A). Single signals were obtained in the analysis of 5` ends of RNAs derived from heterologous transcription assays. An extension reaction with RNAs synthesized in vitro from a soybean atp9 template (satp9XR482) detects the guanosine, which is also the first transcribed nucleotide in vivo (Fig. 5B). Signals corresponding to adenosine and guanosine at positions -2 and -1, respectively, are detected in the analysis of in vitro soybean RNA b/c transcripts (Fig. 5C). The difference of 1 or 2 nucleotides in comparison to the 5` end mapped for in vivo transcripts might be due to a slightly altered migration behavior of the cDNA molecule compared with the sequencing products.


Figure 5: Primer extension analysis of in vitro synthesized RNA derived from transcription reactions using pea mitochondrial protein extract and homologous and heterologous DNA templates. The primer extension reaction was performed on in vitro transcripts recovered from a polyacrylamide gel. To determine the exact 5` ends of the run-off transcripts the extension products (pex) were coelectrophoresed with sequencing reactions performed with the same oligonucleotides as in the primer extension reactions. For easier interpretation, the sequencing reactions have been labeled in the inverted order CTAG to show the sequence of the sense strand given in the right margins. Signals of the extension reactions are indicated by horizontal arrows; 5` ends determined from isolated in vivo transcribed RNAs are indicated by bent arrows (Brown et al., 1991; Morikami and Nakamura, 1993). The boxed sequence represents the conserved nonanucleotide sequence element of mitochondria from dicotyledonous plants. A, primer extension analysis of 5` ends of in vitro transcripts derived from KpnI-linearized DNA template patp9SK630 containing the pea atp9 promoter. B, analysis of in vitro transcription products obtained with KpnI-linearized soybean atp9 template satp9XR482. C, determination of the 5` ends of run-off transcription products obtained in transcription reactions with KpnI-digested RNA b/c template (KM2F2EH) from soybean mitochondria.



Despite the ambiguous results obtained in the investigation of the pea atp9 transcripts, the primer extension analysis of heterologous in vitro transcripts derived from soybean templates indicates the capability of the pea mitochondrial lysate to accurately initiate transcription at the start sites determined for in vivo transcribed RNA.

Deletion Analysis of the Pea atp9 Promoter

Analysis of the pea atp9 promoter focused on the contribution of the region upstream of the transcription start site and on the role of the conserved nonanucleotide as a functional element in mitochondrial promoters of dicot plants. For this purpose a series of constructs with progressively deleted sequences upstream of the respective transcription start site were generated from clone patp9SK630 (Fig. 6A). The DNA templates were linearized with KpnI and tested for their ability to direct transcription initiation. A 355-nt RNA species is expected upon correct initiation at the atp9 promoter on this template (Fig. 1A). Transcription product analysis shows that deletion of sequences upstream of nucleotide -25 has no significant effect on the initiation process in vitro (Fig. 6B, Deltapatp9 -67 to Deltapatp9 -25). Deletion of additional sequences reduces specific transcription initiation almost completely (Fig. 6B, Deltapatp9 -7 and Deltapatp9 +2). This indicates that sequences required for transcription initiation in vitro are clustered immediately upstream of the transcription start site within the 25 nucleotides that are directly upstream. Although these essential sequences contain the conserved nonanucleotide, the conserved sequence element alone is not sufficient to efficiently promote transcription, because deletion of the sequence between the nonanucleotide and nucleotide -25 reduces the rate of in vitro transcription almost completely (Fig. 6B, Deltapatp9 -7).


Figure 6: Deletion analysis of the nontranscribed region upstream of the pea atp9 transcription start site. A, using PCR techniques, sequences were successively removed from clone patp9SK630. Designations of the constructs used in the transcription assays are given in the left margin. Bold lines illustrate mitochondrial sequences. Vector sequences are given as thin lines. Numbers indicate nucleotide positions with respect to the transcription start site (+1, shown as bent arrows). The conserved nonanucleotide motifs are indicated by black boxes. Restriction sites are indicated for KpnI (K), SfuI (Sf), and BamHI (B). B, part of a PhosphorImager print of the transcription products separated on a polyacrylamide gel. The size of the run-off products obtained upon in vitro transcription of KpnI-linearized templates (designations given in the top part) is shown in the left margin. Sizes of coelectrophoresed length standards (M) are shown at the right hand side. Faint transcription products are observed with Deltapatp9 -7 that are too weak to show up in print.




DISCUSSION

In Vitro Transcription System for Mitochondria of Dicot Plants

In vitro transcription systems have been successfully established for mitochondria of a number of different organisms including the two monocot plant species wheat and maize (Edwards et al., 1982; Walberg and Clayton, 1983; Bogenhagen and Yoza, 1986; Kennell and Lambowitz, 1989, Hanic-Joyce and Gray, 1991; Rapp and Stern, 1992). One of the major difficulties during the development of an in vitro transcription system for dicot plants was encountering the identification of a plant species amenable as a source for the isolation of large amounts of transcriptionally active mitochondria. We have now established such a system for pea mitochondria that allows the investigation of various features of the transcription initiation process in dicot plants.

Fractionating pea mitochondrial extracts on a MonoQ column showed that the formation of transcription initiation complexes is favored by the enrichment of RNA polymerase and potential transcription factors in an optimal stoichiometric proportion in the 250 mM KCl elution step. The vast majority of nonspecific transcription activity (i.e. RNA polymerase separated from specificity factors) elutes with the 400 mM KCl fraction. Nonspecific transcription activity is additionally enhanced by the contamination of this fraction with endogenous mtDNA, which elutes from the MonoQ column at KCl concentrations higher than 300 mM. Cofractionating intact mtDNA-RNA polymerase complexes possibly also contribute to background activity. High molecular weight transcripts detected in all transcription assays are most likely due to nonspecific initiation events. Similar transcripts were also observed in other in vitro transcription systems and are attributed to initiation events at ends or nicks of the linear DNA templates (Hanic-Joyce and Gray, 1991; Rapp and Stern, 1992).

Further purification of the active protein fractions supplemented by DNA binding assays will be necessary to characterize the individual protein components involved in the transcription initiation process.

Accuracy of the Pea Mitochondrial Transcription System

Every in vitro system should clearly and accurately represent the in vivo process investigated. In this respect the precise initiation in an in vitro transcription system should be identical with the transcription start site determined in vivo.

In the pea system described here, transcription is indeed initiated precisely on the soybean atp9 template (Fig. 5B). With the soybean RNA b/c template, a 5` end is detected that is very close to the 5` transcript terminus determined by an in vitro capping analysis (Fig. 5C). We assume that the differences between 5` ends of in vitro run-off transcripts, which are scattered over several nucleotides, and of in vivo pea atp9 mRNAs, where a single G is detected as a 5` terminal nucleotide, are derived through intrinsic problems of the primer extension experiments (Fig. 5A). The in vitro synthesized pea atp9 transcripts, which are recovered from the gel and used as RNA templates in the extension analysis, are always detected as a single sharp signal as seen for example in Fig. 1B. A series of transcripts with different 5` ends ranging over 10 nucleotides, as indicated by the primer extension analysis, should rather appear as an expanded signal separated in a 5% polyacrylamide gel (Fig. 1B).

Because a single signal consistent with the result obtained by Morikami and Nakamura(1993) (indicated by a bent arrow in Fig. 5A) is always detected in primer extension experiments with isolated in vivo pea mtRNA (data not shown), we conclude that the artificial length and composition of the in vitro run-off transcripts might be responsible for the scattered termini in the primer extension experiments most likely by a disadvantageous back folding.

Competence of the Pea in Vitro Transcription System for Promoters of Various Dicot Plants

Beyond the competent transcription of homologous templates and templates from another legume, soybean, the pea system initiates correctly also at promoters from the more distantly related dicot Oenothera (Onagraceae). This includes, in addition to the initiation sites of cox2 and atp1, the promoter for a tRNA gene. The correct recognition of these promoters now provides experimental evidence for a more general conservation of promoter structures for at least two types of RNAs in mitochondria of dicot plant species. The dissemination of the conserved nonanucleotide motif at 5` ends of transcripts in other dicot species extends this broad significance, although evidence for genuine promoters in these species is still lacking (Moon et al., 1985; Young et al., 1986; Rothenberg and Hanson, 1987). However, in dicot plant mitochondria, transcription seems to be somehow additionally discriminatory, because the Oenothera 18 S rRNA promoter, although identical with respect to the conserved nonanucleotide, is not recognized by the pea in vitro system (data not shown).

The Conserved Nonanucleotide Motif Is a Core Element of the Dicot Plant Mitochondrial Promoters

Almost all DNA templates carrying the conserved nonanucleotide motif (5`-CRTAAGAGA-3`) were recognized in vitro even on heterologous template DNA. Because this motif constitutes the only sequence element conserved in the active promoter regions, the in vitro transcription analysis strongly supports the conserved nonanucleotide motif to represent the core element of a mitochondrial promoter structure in various dicot plant species. This is confirmed by the deletion studies, in which only deletion of sequences immediately upstream of the transcription start point (i.e. 25 nucleotides preceding the transcription start site) significantly reduces initiation activity. Although deletion of the conserved nonanucleotide abolishes initiation completely (Fig. 6B, Deltapatp9 +2), a small amount of transcriptional activity remains in a construct with sequences up to position -7 (Fig. 6B, Deltapatp9 -7, detectable on the original x-ray film). The strongly reduced activity suggests an extended promoter structure, where the conserved nonanucleotide motif functions as a core element indispensable for transcription initiation and additional upstream sequences amplify initiation efficiency.

A comparison of sequences located between nucleotide positions -25 and -8 reveals a high content of adenosine-thymidine base pairs (67-83%) with almost 100% of the AT base pairs concentrated between nucleotide positions -13 to -9. This AT-box and a conserved adenosine nucleotide at position -are the most remarkable features within the critical upstream region (Fig. 7). The presence of the AT-rich region is most likely responsible for enhanced melting of the DNA strands during the transcription initiation process.


Figure 7: Alignment of sequences containing the promoters transcribed by the pea mitochondrial extract. Sequences critical for the transcription of the pea atp9 promoter were compared with other promoters recognized by the pea lysate and the maize atp1 promoter, respectively (Rapp et al., 1993). Nucleotide positions are given with respect to the first transcribed nucleotide (position +1, underlined). Highly conserved sequences in the dicot promoter regions are boxed and designated AT-box (for AT-rich box) and CNM (for conserved nonanucleotide motif). The 5`-YRTA-3` motif common to dicot and monocot promoter sequences is underlined. Sequence elements critical for the transcription of the maize atp1 promoter are the ``upstream domain'' (boxed) and the core element (underlined) of the central domain. Adenosines and thymidines are shown in bold and the adenosine-thymidine contents of sequences between nucleotide position -25 and -8 are given in the right margin. The distances between the first transcribed nucleotide, the center of the AT-box at position -10, and the conserved adenosine at -20 locate these elements to the same side of the DNA-helix, suggesting their recognition by a protein factor(s). Although the conserved nonanucleotide motif element is essential for transcription, the AT-rich box is found to be required to raise transcription initiation to significant levels.



This model is similar to the maize atp1 promoter structure (Rapp et al., 1993), although the core element in dicot plants appears to be extended and much better conserved than their counterpart in monocots. Additional detailed deletion and mutagenesis studies will define the exact promoter structure and requirements in mitochondria of dicot plant species.

Do Other Types of Promoters Exist in Mitochondria of Dicot Plants?

In addition to the conserved nonanucleotide promoters, several transcription initiation sites have been described that show almost no similarity to this motif or to each other. An example is the 26 S rRNA gene, whose transcription remains controversial in dicot plant mitochondria (Binder et al., 1994). Although transcription of this gene in potato is initiated at the mature 5` end of the rRNA, the transcription initiation site of the Oenothera 26 S rRNA gene remains unclear. A test of both potato and Oenothera 26 S rRNA templates in the pea in vitro system failed to show any significant specific transcription activity. In the pea in vitro system, only promoters containing the conserved nonanucleotide appear to be transcribed. The transcription start site for RNA e in soybean does not contain the nonanucleotide motif and is not recognized in vitro. Although located on the same template as the promoter for RNA b/c (Fig. 3A, KM2F2EH), only RNAs with sizes that indicate initiation at the RNA b/c promoters were detected in these assays (Fig. 3B). Additional slightly smaller RNAs observed with the KpnI-linearized template (Fig. 3B, lane 1) are most likely degradation products, because these shorter transcripts are not present in the reaction with the HindIII-cleaved template. This observation suggests that mitochondria of dicot plants exploit different modes of transcription initiation using alternative promoter structures.

Only a few of these alternative promoters have been identified and so far no similarity could be detected between their primary structures. These nonconserved promoters represent probably gene- and/or species-specific single copy promoters. Because in animal and yeast mitochondria replication is primed by short RNAs initiated at promoter sequences, a similar function might be attributed to some of these alternative promoters in plants (Schmitt and Clayton, 1993; Xu and Clayton, 1995). Much more data about these promoters in a single plant species are necessary to see whether different types or classes of promoters are indeed present in plant mitochondria and how they are recognized.


FOOTNOTES

*
This work was supported by grants from the Deutsche Forschungsgemeinschaft and the Bundesministerium für Forschung und Technologie. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 49-30-83000750; Fax: 49-30-83000736; Binder{at}RZ-Berlin.MPG.DE.

(^1)
The abbreviations used are: PCR, polymerase chain reaction; nt, nucleotide(s).


ACKNOWLEDGEMENTS

We are very grateful to Atsushi Morikami and Gregory Brown for providing pea and soybean plasmid clones, respectively, and to Martina Gutschow for her skilled technical assistance. We also thank David Stern and Bill Rapp for very helpful hints during the development of the transcription system.


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