ABSTRACT
The expression of two mitochondrial gene clusters (
orf87-nad3-nad1/A
and
orf87-nad3-rps12
) was studied in
Nicotiana sylvestris
. 5
'
and 3
'
termini of transcripts were mapped by primer extension and nuclease S1
protection. Processing and transcription initiation sites were differentiated
by
in vitro
phosphorylation and capping experiments. A transcription initiation site, present in both gene clusters, was found 213 nucleotides upstream of
orf87
. This promoter element matches the consensus motif for dicotyledonous mitochondrial promoters and initiates
run-off transcription in a pea mitochondrial purified protein fraction. Processing sites were identified 5
'
of
nad3
,
nad1/A
and
rps12
respectively. These results suggest that (i) the expression of the two cistrons is only controlled by one duplicated promoter
element, and (ii) multiple processing events are required to produce
monocistronic
nad3
,
nad1/A
and
rps12
transcripts.
In comparison with mammals and fungi, the expression of mitochondrial (mt) genes
in plants and especially the regulatory role of promoter regions is still
poorly documented. In mammals, the mt genome is small as compared with higher
plants (16 versus 200-2000 kb). Transcription is initiated from two promoters located in the non coding D-loop region and produces essentially two polycistronic primary transcripts, each corresponding to one strand of the circular genome (
1
). In yeast, several polycistronic transcripts have been characterized and the 20 identified transcription initiation sites
share a nonanucleotide consensus element having the capability to initiate
in vitro
transcription (
2
). In higher plants, mt genes are dispersed throughout the genome and are often
separated by large non coding regions. In contrast with the coding sequences,
gene flanking regions are poorly conserved (
3
),
making each investigation on gene expression a particular case. Another
peculiarity is the presence
,
for some genes, of multiple transcription initiation and/or processing and
termination sites frequently resulting in complex transcription patterns (
4
,
5
). Nevertheless, the recent development of
in vitro
transcription systems for plant mitochondria has allowed rapid progress for the
understanding of promoter structures. In the maize
atp1
gene, an 11 nucleotide (nt) promoter consensus was identified by deletion
experiments (
6
). Linker scanning and site-directed mutagenesis further established the fundamental role of a 12 nt
central domain surrounding the initiation site (
7
). In dicots, a consensus sequence of 29 nt with a highly conserved core of 9 nt
was reported in
Oenothera berteriana
(
8
,
9
). A 20-30 nt more precise consensus sequence was defined by
in vitro
transcription at the initiation sites of several mt dicotyledonous genes coding
for mRNAs and tRNAs (
10
). From these studies, only a short motif of 4 nt (CRTA) appears to be common to
the mono- and dicotyledonous mt promoter sequences (
3
). Characterization of transcription initiation sites in additional genes of different species will be useful to describe more
accurately the nature of plant mt promoters.
Although many plant mt genes are expressed independently, some coding sequences
are co-transcribed in the form of long multicistronic primary transcripts. Some gene clusters, such as
rrn18
-
rrn5
,
nad3-rps12
or
rps3-rpl16
, have been found in virtually all species investigated so far (
11
). However most of the gene clusters appear to be specific of one or a few
species, as for example
atp9
-
rps13
in tobacco (
12
),
coxIII
-
orf25
(
13
) and the
rps3-rpl16-nad3-rps12
units in rice (
14
). Northern and RT-PCR experiments confirmed co-transcription of the associated genes, but many questions are still
to be addressed in concern of the expression and functional significance of the
gene clusters.
In the solanaceous species
Nicotiana sylvestris,
an orf called
orf87
and the
nad3
gene are present in two mt genomic regions. In the first, they are located
upstream of
rps12
while in the second, the exon A of
nad1
(named
nad1/A
) has been identified downstream of
nad3
(
15
). Previous RT-PCR experiments showed that the coding sequences in the two different gene clusters are co-transcribed (
15
). In this study, transcription of both genomic regions in
N.sylvestris
mitochondria was investigated by mapping transcription initiation, processing and termination sites.
The fertile
N.sylvestris
line (T) is a botanical wild-type line provided by the Institut des Tabacs (SEITA, Bergerac, France) and has been
maintained by self pollination for >15 generations. Plants were grown in
greenhouses under 16 h day length at 24oC (day) and 17oC (night). Pea seedlings (
Pisum sativum
L., var. Progress No.9 and var. Lancet) used in run-off transcription assays were grown in the dark for 7 days before extraction of mitochondria.
Plasmid clones KS5.3 and KS7 contain the 5.3 and 7 kb
Sac
I restriction fragments of
N.sylvestris
mt DNA, that are respectively
carrying
the
orf87-nad3-nad1/A
and
orf87-nad3-rps12
cistrons (
15
). Sequences corresponding to both loci are available in the DDBJ/EMBLGenBank
library under accession numbers X96741 and X96742. Clones 1-3 (Figs
3
A and
4
A) were obtained by subcloning three PCR-products into the vector pUC18 according to the `SureClone ligation kit'
protocol (Pharmacia) and were maintained in the
Escherichia coli
DH5[alpha] host strain. These PCR products, amplified using KS5.3 as template, were
respectively a 1.2 kb fragment, spanning the
orf87
upstream region (primers O2 and O6, see primers section), a 729 bp fragment
corresponding to the
orf87-nad3
intergenic region (primers O7 and O8) and a 230 bp fragment, spanning the
nad3
-
nad1/A
intergenic region (primers O4 and O9). PCR experiments were performed in a
final volume of 25 [mu]l: 20-50 ng of DNA template were mixed with 50 pmol of each primer, 0.2 mM
of each dNTP, 1 U of
Taq
DNA polymerase (Appligene) and 2.5 [mu]l of the 10*
Taq
polymerase buffer supplied by the manufacturer. Each of the 30 cycles consisted
of 1 min at 92oC, 2 min at the primer annealing temperature and 1 min at 72oC.
Clone 4 (Figs
3
A and
4
A) and clone 5 (Fig.
5
) contain the
nad3-rps12
intergenic region and part of the
rps12
coding sequence, and correspond to the 115 bp
Spe
I-
Xho
I and to the 370 bp
Spe
I-
Eco
RI subfragments of KS7 respectively. These restriction fragments were subcloned
into the vectors pBluescript KS+ or SK+ (Stratagene) following standard
procedures (
16
).
Nicotiana sylvestris
mt were isolated from young leaves by differential centrifugation and were
purified on discontinuous sucrose gradient, as described elsewhere (
17
). Mitochondria were lysed in 10 mM Tris-HCl pH 7.5, 10 mM MgCl
2
, 1% (w/v) SDS, followed by extraction with 1 vol of water-saturated phenol. Large mt RNAs were isolated by LiCl precipitation (
18
).
DNA sequencing was performed using the T7 sequencing kit (Pharmacia). DNAs were fractionated in 0.8% non denaturing agarose gels, and were transferred on Hybond N membrane according to the manufacturer's
instructions (Amersham).
5'-end-labelling of primers (10 pmol) was performed using 10 U of T4
polynucleotide kinase (Gibco-BRL) and 5 [mu]l of [[gamma]-
32
P]ATP (3000 Ci/mmol) according to (
16
).
100 000 c.p.m. of each labelled primer were co-precipitated with 50 [mu]g of
N.sylvestris
total mt RNAs in ethanol for 20 min at -80oC. After a 20 min centrifugation at 15 000
g
, the pellet was rinsed in 70% ethanol and resuspended in 20 [mu]l of hybridization buffer (0.4 M NaCl, 10 mM PIPES pH 6.4, 1 mM Na
4
EDTA). After heating for 15 min at 70oC, hybridization of the labelled primers to mtRNAs was achieved by a 3-4 h incubation at 42oC. Primer extension was carried out in a final volume of 200 [mu]l with 40 [mu]l of 5* Mu-MLV buffer (250 mM Tris-HCl pH 8.3, 375 mM KCl, 50 mM DTT
and 15 mM MgCl
2
), 200 U of reverse transcriptase (Mu-MLV, Gibco-BRL), 10 [mu]l of each dNTP (10 mM) and 125 [mu]g/ml actinomycin D. After 1 h of incubation at 37oC, RNase A (20 mg/ml) was added for 30 min at 37oC. The DNA-RNA duplexes were ethanol precipitated,
denatured by heating 10 min at 70oC, analyzed in a 6% denaturing polyacrylamide gel and autoradiographed. The sequence of the M13mp19 plasmid (obtained
with the M13 universal primer) was used as marker for product size estimation.
To map 5' (Fig.
1
C) and 3' transcript termini (Fig.
2
), nuclease S1 experiments were performed as described in (
19
). Synthesis of the single-stranded uniformly labelled probe in Figure
1
C was performed using the subcloned
Spe
I-
Apa
I restriction fragment of KS5.3 (containing the
nad3
-
nad1/A
intergenic region and part of
nad1/A
coding sequence) as template and the primer O4 (complementary to the 5' coding region of
nad1/A
). This probe was linearized at its 5' end with
Spe
I. In Figure
2
, the probes corresponding to the
nad1/A
downstream region were synthesized using the subcloned
Apa
I-
Pst
I and the
Pst
I-
Kpn
I fragments of KS5.3 as templates and the M13 universal primer. The two probes
were linearized at their 5' end with
Dra
I and
Pst
I respectively. Product sizes were determined in comparison with the sequence of
M13mp18 plasmid.
In vitro
capping and RNase protection experiments (Fig.
3
) were carried out according to the protocol described in (
20
). Protected products were run in a 6% denaturing polyacrylamide gel with the
standard M13mp18 sequence.
Nicotiana sylvestris
mtRNAs (50-75 [mu]g) were labelled in a final volume of 40 [mu]l containing 4 [mu]l of 10* polynucleotide kinase buffer, 40 U of polynucleotide
kinase (Gibco-BRL), 80 U of RNase Inhibitor and 100 [mu]Ci [[gamma]-
32
P]ATP (3000 Ci/mmol). The reaction was incubated 1 h at 37oC and samples were treated as the capped RNAs.
In vitro
run-off transcription assays were performed as in (
10
). Transcription products were electrophoresed in a 5% denaturing polyacrylamide gel in parallel with a labelled pGEM DNA marker (Promega). The satp9XR482 clone containing the soybean
atp9
gene promoter region and linearized with
Kpn
I (
10
) was used as a positive control.
Positions of the primers given into brackets refer to the sequences of KS5.3
(DDBJ/EMBL/GenBank no.
X96741) and KS7 (X96742). O1, O2, O3, O6, O7, O8 and O9 coordinates refer to
either X96741 or X96742; O4 coordinates refer to X96741 and O5 to X96742. The
position 0 corresponds to the start codon of the
orf87
coding sequence. Primers O1, O2, O3 and O5 were used in primer extension
analysis (Fig.
1
A, B and D) and primer O4 in nuclease S1 protection (Fig.
1
C). Primers O2, O4, O6, O7, O8 and O9 were used in order to obtain the PCR
subfragments of KS5.3.
O1 (-142/-159)
5'-TTTTTATTATGATTGGGC-3'
O2 (51/34)
5'-GCATGACCAGAAGAATTG-3'
O3 (762/745)
5'-GGCGTTTTCCTGGCTTAG-3'
O4 (1470/1453)
5'-AACAGCTATGTACATTTT-3'
O5 (1322/1303)
5'-GATTTTTTGTAGGCATCGCT-3'
O6 (-1206/-1189)
5'-GAGGTCCTCTCCTTACAG-3'
O7 (179/196)
5'-GCTTGCTAACTCTTGGAT-3'
O8 (907/890)
5'-GACATCACCACAGAAAAC-3'
O9 (1259/1276)
5'-ACCACTAGTGAGAGGGCA-3'
In the fertile line of
N.sylvestris
, we previously described two mt gene clusters, the
orf87-nad3-nad1/A
and the
orf87-nad3-rps12
cistrons, localized on two
Sac
I restriction fragments of 5.3 and 7 kb respectively (
15
). The sequence data of both genomic regions (DDBJ/EMBL/GenBank nos X96741 and
X96742) revealed that the 60 first nt of
orf87
are similar to several plant mt chimeric orfs (
15
). In a first step towards identification of a potential promoter element, we
sequenced the clone 1 containing the 1.2 kb upstream region of
orf87
(data not shown). Sequencing data confirmed previous
Southern hybridization and restriction mapping indicating that the
orf87
upstream region is identical in both gene clusters. Moreover, strong similarity
was found between the upstream region of
N.sylvestris
orf87
and the upstream regions of other mt genes: the proximal 128 nt showed 98% identity with the 5' regions of
rps13
and
atp6
genes in tobacco (
12
,
21
) and partial similarity with the sequences upstream of the
Petunia S-pcf
chimeric gene (
22
), of the
Oenothera
and sunflower
orfB
(
23
,
24
) and of
Brassica napus
orf158
and
orf224
(
25
,
26
).
The 5' ends of transcripts were mapped using primer extension and nuclease S1
experiments, which allow detection of both initiation and processing sites.
Primer extension was used to map transcript extremities in the upstream regions
of
orf87
(primers O1 and O2; Fig.
1
A), of
nad3
(primer O3; Fig.
1
B) and
rps12
(primer O5; Fig.
1
D). Nuclease S1 protection was used to map 5' transcript ends in the upstream region of
nad1/A
(primer O4; Fig.
1
C). Primer O2 gave abundant and large products, whose sizes were difficult to estimate but no signal was visible between the
orf87
start codon and the
Apa
I site (data not shown). Primer O1 spanning the
Apa
I site yielded two main signals corresponding to products of 72 nt (site S1:
position -213 with regards to the ATG codon of
orf87
) and 35/40 nt (site S2: position -175/-180). Another 5' RNA extremity was mapped 481/484 bp upstream of
nad3
with primer O3 (site S3: position 418/421). On the other hand, nuclease S1
protection, using the O4-
Spe
I single-stranded labelled probe spanning the
nad1/A
upstream region, gave two major signals. One of 208 nt corresponded to the size
of the probe which is thus fully protected by
nad3
-
nad1
/
A
co-transcripts. A second shorter product of 80-85 nt indicated a 5' transcript end located 65/70 bp upstream of
nad1
/
A
open reading frame (site S4: position 1386/1391). Primer extension using primer
O5 located near the
rps12
start codon yielded a major product of 46/52 nt corresponding to a 5' site located 30/36 bp upstream of
rps12
(site S5: position 1271/1277).
3' termini of the
orf87-nad3-nad1/A
transcripts were mapped by nuclease S1 protection. Uniformly labelled probes corresponding to the
Pst
I-
Apa
I and
Kpn
I-
Pst
I subfragments downstream of
nad1
/
A
were synthesized, restricted with
Dra
I and
Pst
I respectively and hybridized with mtRNAs. After nuclease S1 treatment, two products of 193 nt
(
Apa
I-
Pst
I template; Fig.
2
A) and 259 nt (
Pst
I-
Kpn
I template; Fig.
2
B) were obtained corresponding to sites located 188 bp (site T2: position 2373)
and 785 bp (site T1: position 2970) downstream of the
nad1/
A stop codon. This 5' and 3' transcript termini mapping correlates well to the transcription
patterns obtained in northern experiments using
orf87
,
nad3
and
nad1/A
(
15
). Indeed the 3200 and 2700 nt RNA species detected with the three probes could
correspond to the sizes expected for large co-transcripts beginning at site S1 and ending at termination sites T1 and T2
respectively.
Primary mitochondrial RNAs are not capped during
in vivo
maturation and can be capped
in vitro
using guanylyltransferase. This enzyme recognizes these transcripts with free
tri- or biphosphate 5' extremities, but not processed mtRNAs with a monophosphate extremity. In order to get preliminary information on the localization of potential transcription initiation sites,
in vitro
cap-labelled total mtRNAs were hybridized to Southern blots carrying different regions covering each gene cluster, i.e. clones 1-4 (corresponding to the upstream regions of
orf87, nad3, nad1/A
and
rps12
respectively) and
the
Sac
I-
Apa
I and
Sac
I-
Bam
HI restriction digests from KS5.3 and KS7 (Fig.
3
A). A signal was observed with clone 1 containing the sites S1 and S2, but not
with clones 2, 3 and 4 (Fig.
3
B). Hybridization was also detected with the 1 kb
Sac
I-
Apa
I region upstream of
orf87
and the
Sac
I-
Bam
HI fragment of 1.75 kb containing the sites S1 and S2 (Fig.
3
C). Analogous results were obtained with the
Sac
I-
Apa
I and
Sac
I-
Bam
HI digests of KS7 (Fig.
3
C). Taken together, these data suggest that in both gene clusters, transcription
initiation sites are located in the upstream region of
orf87
and that no additional initiation site exists.
As a confirmation, capped mtRNAs were hybridized to an antisense single-stranded RNA complementary to clone 1 (linearized at the
Hin
dIII site of the plasmid multiple cloning site) and the RNA-RNA duplex was submitted to RNase digestion. A protected product of 225
nt was obtained (Fig.
3
D, lane 2) which corresponds exactly to the site S1, located 213 nt upstream of
orf87
. This site thus seems to function as a transcription initiation site.
In order to confirm the capping data, phosphorylation of mtRNAs was performed.
Since primary transcripts have bi- or triphosphate extremities, only the processed mtRNAs with 5' monophosphate extremities will be labelled by a polynucleotide
kinase activity. Phosphorylated mtRNAs were hybridized to Southern blots of
clones 1-4 and all clones except clone 1 displayed a signal (Fig.
4
A). In the
Sac
I-
Apa
I and
Sac
I-
Bam
HI restriction digests of KS5.3 and KS7, all fragments except the 1 kb
Sac
I-
Apa
I and the 1.75 kb
Sac
I-
Bam
HI fragments hybridized to the phosphorylated mt RNAs (Fig.
4
B). Phosphorylation experiments thus confirm the
in vitro
capping analysis, suggesting that the site S1 is an initiation site and that
S3, S4 and S5 are processing sites.
Co-transcription of mitochondrial genes encoding polypeptides involved in
different functional pathways and present in different amounts creates unique
regulatory difficulties. The mt
orf87- nad3-nad1/A
and
orf87-nad3-rps12
cistrons in
N.sylvestris
were investigated as examples of such units. In these gene clusters,
nad1
and
nad3
encode subunits of the NADH: ubiquinol oxidoreductase involved in the mt respiratory process, while
rps12
encodes a polypeptide of the small ribosomal subunit, known to be essential in the translational apparatus (
28
). Whereas the
orf87-nad3-nad1/A
cistron appears to be specific of the
Nicotiana
genus (
15
), it is noteworthy that the
nad3
-
rps12
association is conserved in all higher plants tested so far. Such conserved
organization is also observed for the
rrn18
-
rrn5
and
rps3-rpl16
gene clusters which appear to have a functional significance, as the associated
genes are all involved in the mitochondrial translational apparatus. The
conservation of the
nad3
-
rps12
cluster could be explained by structural arguments, i.e. no homologous
recombination has occurred within the
nad3
-
rps12
intergenic region during plant evolution (except in the
Nicotiana
genus;
15
), because of its small size or uniqueness of its sequence. Another possibility is that this gene
association displays a functional significance in plant mitochondria, but neither quantitative nor functional correlation between
nad3
and
rps12
transcripts or polypeptides has been reported to date.
Primer extension experiments revealed two signals (S1 and S2) in the upstream
region of
orf87
in both gene clusters.
Sequencing of this region showed that a 128 bp sequence located upstream of
orf87
displays homology with the upstream regions of other mt genes and orfs. Such a
conservation suggests that it might play an important role in the expression of
these coding sequences. Since the sites S1 and S2 are located outside this
conserved region (as they were found 213 and 175 bp upstream of
orf87
respectively
)
we assume that, if the conserved region plays a role, it may be involved in the
post-transcriptional maturation of the transcripts or in the translational
processes. The nature of the site S2 is not clear, since taken together all
experimental data show that it is neither an initiation site nor a processing
site. Furthermore, sequence analysis of this site did not reveal the presence
of any potential promoter element. Since the S2 signal was repeatedly found, we
can suggest either that a secondary structure leads to the premature stop of
the reverse transcriptase or, that the primer O1 could partially prime another
transcript and thus allow the synthesis of the observed short reverse
transcription product.
In vitro capping and transcription experiments showed that the site S1 is a
transcription initiation site. Sequence of the region surrounding this site
fits the consensus proposed for dicotyledonous mt promoters by comparision of
rrn
,
trn
but also
atp
and
cox
genes from
Oenothera
, pea and soybean (
36
):
Few data are available concerning mt gene promoters in solanaceous species, as
capping experiments have only been performed for the
S.tuberosum trnS-trnF-trnP
cistron and for the
atp9
and
rrn26
genes
(
20
,
29
,
30
)
.
In these cases, the transcription initiation sites do not display similarity
with the dicotyledonous consensus sequence. We show here that a promoter with
this consensus exists in a solanaceous species and that it is used for
transcription of both
nad
and
rps
genes. Moreover this promoter element is able to initiate transcription in a
pea
in vitro
system. Among promoter regions described to date in dicots, only the 20-30 bp consensus sequence surrounding the initiation site and adjacent AT-rich regions have been shown to play a role in mitochondrial
transcription initiation, but no additional activator or inhibitor element has
yet been reported (
3
,
7
,
10
). Nevertheless such regulatory elements are likely to be present since run-on transcription assays showed that genes displayed different
transcription rates in plant mitochondria (
28
,
31
).
In vitro
transcription systems, like the pea one, are thus expected to allow the
investigation of potential 5' and/or 3' regulatory motifs, by comparing genes with different transcription rates or tissue-specific expression (
32
,
33
).
In contrast with wheat or rice, the expression of
nad3
and
rps12
genes in
N.sylvestris
appears to be controlled exclusively by the
orf87
promoter region. In wheat, a pseudo-tRNA ([Psi]-tRNA
Phe
) is located upstream and co-transcribed with the
nad3-rps12
unit (
34
). Two orfs located downstream of
rps12
also belong to the cistron (
35
). Primer extension experiments detected at least two putative transcript
extremities in the 5' region of
nad3
, one upstream of the [Psi]-tRNA
Phe
gene and a second within the [Psi]-tRNA
Phe
coding sequence. Unfortunately capping experiments were not performed and the function of these sites remains unclear. In rice,
nad3
and
rps12
are located downstream of and are co-transcribed with the
rps3-rpl16
gene cluster (
14
). Capping experiments revealed two alternative promoters, one upstream of
rps3
leading to a large polycistronic transcript and another allowing independent
transcription of the
nad3
-
rps12
cluster.
The mechanisms of RNA processing in plant mitochondria are still little
understood (
36
). Some data are available concerning tRNA and rRNA processing (
3
,
37
) but very little is known about mRNA maturation. In
N.sylvestris
,
we have shown that the 5' transcript termini detected upstream of
nad3
(site S3),
nad1/A
(site S4) and
rps12
(site S5) are assumed to be processing sites since they yielded no signal in either capping or
in vitro
transcription experiments. Furthermore, complementary hybridization of
in vitro
phosphorylated mtRNAs as probe to DNA fragments containing these sites supports
the idea that they indeed correspond to processed RNA extremities. The sequences at and around these sites are divergent and do not display any similarity with conserved sequences of
published processing sites (
38
,
39
). In addition, two different transcript 3' termini (called T1 and T2) were localized in the
nad1/A
downstream region. These 3' ends are also very divergent and they do not show primary sequence
similarity with termination or processing sites of mt genes from other species
(
40
-
42
). According to the divergence of the primary sequences near all these sites,
even within the same species, it is very difficult to speculate about the mechanism and specificity of processing or termination. Nevertheless 3' terminal secondary structures have been proposed either to characterize
transcription termination as in bacteria (
43
) or to play a role in mRNA processing or transcript stabilization as in the
chloroplast (
44
). In this study, a stem-loop structure could be folded at the site T2 only (Fig.
2
). The strong intensity of nuclease S1 signal suggests that transcripts ending
at this site can accumulate in
N.sylvestris
mitochondria and supports the idea of a functional importance of such secondary
structure in tobacco mitochondria. On the other hand, previous northern
experiments (
15
) revealed strong signals for transcripts assumed to be ending at the processing
sites S3, S4 or at the termination site T1. Since these sites do not display particular secondary structures, it may be proposed that alternative factors and/or other motifs could play a
role in mRNA processing and stabilization, such as RNA binding proteins which
have already been found in the chloroplast (
45
).
As no initiation site was identified within the different intergenic regions, we
assume that
nad3
,
nad1/A
and
rps12
are exclusively expressed as polycistronic cotranscripts in
N.sylvestris
. Similar results were obtained for the
nad3-rps12
association in wheat and rice (
35
,
13
) as well as in the maize co-transcribed
rrn18
and
rrn5
genes (
4
). Nevertheless posttranscriptional maturation of the primary transcripts
encoded by the
nad3-rps12
gene clusters appears to differ between species. Indeed our results suggest
that, in
N.sylvestris, nad3
and
rps12
monocistronic transcripts can be produced as one processing site was detected
in their intergenic region. In contrast,
nad3-rps12
dicistronic transcripts but no monocistronic RNA were identified in wheat
mitochondria. In this case, the authors reported the presence of a Pribnow
motif between the two genes but no processing site (
34
). Thus, although the
nad3-rps12
gene association is highly conserved in plants, the flanking co-transcribed sequences as well as the expression patterns differ
significantly between species.
In conclusion, we have shown that in
N.sylvestris
, (i) the expression of two different gene clusters is controlled by a promoter element fitting the dicotyledonous consensus and (ii) the production of the monocistronic transcripts requires multiple processing events of the
large primary transcripts. Such data confirm that, specially for such co-transcriptional units that contain genes belonging to different complexes
or pathways, the post-transcriptional mechanisms are particularly important to regulate the stoichiometry of the transcripts and of the encoded polypeptides in plant mitochondria.
C.L. was supported in part by a short-term fellowship from the European Molecular Biology Organization. C.R. was a Senior Research
Assistant from the Belgian Fonds National de la Recherche Scientifique. We wish
to thank C. Colas des Francs-Small for reading of the manuscript and R. de Paepe for helpful discussions.
*To whom correspondence should be addressed. Tel: +33 1 69 33 64 07; Fax: 33 1
69 33 64 25; Email: chetrit@ibp.u-psud.fr
REFERENCES
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