(Received for publication, April 24, 1995; and in revised form, June 21, 1995)
From the
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.
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.
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.
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.
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).
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).
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.
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.
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 patp9 -7 that are too weak to show up in
print.
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.
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.
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.
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.