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INTRODUCTION |
The established endosymbiont hypothesis describes mitochondria and
chloroplasts as remnants of bacteria-like progenitors, which penetrated
the original eukaryote 1.5 and 1 billion years ago (1). The prokaryotic
origin of the organellar DNAs implies that cis-elements engaged in the
expression of the organellar genetic information might be similar to
the respective control structures of modern bacteria. Such a similarity
is indeed found in chloroplasts, where several components of the
genetic system show a close structural and functional relationship to
their prokaryotic counterparts (2). Many chloroplast promoters for
instance resemble the
10/
35-type promoters of contemporary
prokaryotes. These promoters serve as cis-elements for a largely
chloroplast encoded eubacteria-like RNA polymerase. Another, nuclear
encoded polymerase initiates transcription at a second less well
described class of promoters (3-6). This latter enzyme is of the
bacteriophage-like single subunit type, a class of polymerases that
also includes the RNA polymerases in mitochondria of animals, fungi,
and plants (7-9). These enzymes bind to promoters in close spatial
relationship to transcription initiation sites.
In mitochondria of Saccharomyces cerevisiae a
9-base-pair-long highly conserved cis-element is found at each of the
about 20 transcription initiation sites. This nonanucleotide motif
autonomously supports efficient transcription initiation in
vitro, with enhanced promoter activity depending on the presence
of a purine at position +2 and a pyrimidine at +3. In Xenopus
laevis mitochondria an eight-nucleotide-long cis-element drives
bi-directional transcription initiation at two different locations. A
15-bp1 consensus motif is
sufficient for basal promoter activity in mammalian mitochondria, while
maximal promoter function depends on additional upstream sequence
elements (10, 11).
In vitro capping analyses have identified conserved
sequence elements also at mitochondrial transcription initiation sites in plants. These investigations revealed the tetranucleotide 5'-CRTA-3' to be the only common motif found at initiation sites of both monocot
and dicot plant species (12-16). Functional studies of the maize
atp1 promoter in a homologous in vitro
transcription system confirmed the significance of some of the
conserved sequence features (i.e. 5'-CRTA-3'), while other
segments appear to be not required at least for basic promoter function
in vitro (e.g. G(AT)3-4; Refs. 16
and 17). Further investigations revealed also differences in the basic
structures and requirements between individual promoters. While an
A-rich upstream domain contributes significantly to the maize
atp1 promoter function, such a domain appears not to be
required for the cox3 promoter activity in the same species
(18, 19).
Mitochondrial promoters in dicot plant species display a somewhat
larger conserved sequence element compared with those found in
monocots. Inspection of sequences adjacent to transcription initiation
sites of several bona fide dicot promoters revealed a conserved
nonanucleotide motif (5'(
7)-CRTAAGAGA(+2)-3',
CNM), which covers the first two transcribed nucleotides (20).
Functional studies, however, showed that this motif per se
is not sufficient for the function of the pea atp9 promoter in vitro and that additional sequences are required (21). We have now determined these additional sequence requirements and report
that essential promoter sequences require specific nucleotide identities within an 18-nucleotide region between positions
14 and +4
relative to the transcription initiation site.
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EXPERIMENTAL PROCEDURES |
Template Construction--
5' progressively deleted fragments of
pea atp9, soybean atp9, and Oenothera
rpl5 promoter region were amplified by PCR using Pfu
polymerase in buffer supplied by the manufacturer (Stratagene). PCR
conditions, primer pairs, and DNA templates were as follows. Pea
atp9 promoter region (patp9): dp+355 and one of the
oligonucleotides dp-16, dp-14, dp-12, dp-10, and dp-7 using patp9SK630
as template (21); soybean atp9 promoter region: Rev and one
of the oligonucleotides GM-D-7, GM-D-10,
GM-D-12, GM-D-14, and GM-D-16 using
clone satp9XR482 as DNA template (21); Oenothera rpl5
promoter region: OL5-3 and one of oligonucleotides L5/dp-7, L5/dp-10,
L5/dp-13, L5/dp-14, and L5/dp-7 using a
BamHI/HindIII clone comprising the
Oenothera rpl5 gene and its 5'-flanking
sequences.2 DNA fragments
were amplified with 40 cycles of 1 min at 90 °C, 1 min at 50 °C
(pea atp9), 40 °C (soybean atp9), and 45 °C
(Oenothera rpl5), respectively, and 1 min at 72 °C. PCR
products were ethanol-precipitated and digested with the respective
restriction enzymes and DpnI to remove the template DNA.
After separation in agarose gels DNA fragments were cloned between
BamHI and KpnI (patp9 and satp9) and
BamHI and HindIII (orpl5) restriction sites of
pBluescript vectors.
For substitutions in the 3'-flanking region of the pea
atp9 conserved nonanucleotide motif inverse PCR was
performed with oligonucleotides LS-5-1 and LS-3-1 for clone LS-1 and
LS-5-2 and LS-3-2 for mutant LS-2 on patp9SK630 and patp9SK630
-172 clones as templates, respectively. Partial digestion by
PstI resulted in variations of the inserted sequences. Due
to cloning artifacts during construction of LS-2 an XbaI
site was used for linearization instead of KpnI with
construct LS-1.
Primer pairs TVAT-u and atp9-decad (A/T TM), patp9-1u and patp9-1d
(TM-1), patp9-2u and patp9-2d (TM-2), patp9-3u and patp9-3d (TM-3),
and patp9-2uwt and patp9-2d (TM-2') were used in the inverse PCRs for
transversion of nucleotide identities within the pea atp9
AT-Box, the CNM, and the 3'-flanking sequences, respectively.
Inverse PCRs were exclusively performed with Klentaq polymerase
(CLONTECH) in a buffer supplied by the manufacturer
with 35 cycles under the following conditions: 1 min 94 °C, 1 min
46 °C (TM mutants) and 50 °C (LS mutants), respectively, and 4 min at 68 °C. After PCR the template DNA was removed by digestion
with DpnI, the DNAs were separated on agarose gels and the
respective fragments were ligated at 15 °C for at least 16 h.
All DNA templates used in the experiments described in this report are
controlled by restriction digests and complete sequence analysis.
Sequences of all oligonucleotides used are available on request.
About 18 pmol of each MP oligonucleotide (MP sense:
5'-ATAATAGCATAAGAGAAG-3' and the respective antisense oligonucleotide) were denatured for 30 s at 80 °C, incubated for 20 min at
28 °C, and slowly adapted to room temperature in a total volume of
10 µl. Prior to ligation into the SmaI and
EcoRV sites in pBluescript and the SnaBI site of
a clone containing the pea rps14/cob spacer region, ~9
pmol of double-stranded MP oligonucleotides were concatemerized in the
presence of 12% (v/v) polyethylene glycol, 50 nmol ATP, 1 mM dithiothreitol, and 10 units of T4 DNA ligase. 5 µl of
the concatemerized MP oligonucleotides were then cloned into
pBluescript vectors and a rps14/cob clone in the presence of
0.05 mM ATP, 1 mM dithiothreitol, 11% (v/v)
polyethylene glycol, and 10 units of T4 DNA ligase in a total volume of
15 µl.
In Vitro Transcription Assays--
Enrichment of pea
mitochondrial lysates and in vitro transcription reactions
were performed as described previously (21). Between 23 and 47 µg of
lysate were used in standard assays with 100 ng of template DNA
quantified both photometrically and on agarose gels.
Miscellaneous Methods--
Standard methods in molecular biology
were performed following Sambrook et al. (23). DNA
sequencing was done with Thermo Sequenase fluorescent labeling kits
(Amersham Pharmacia Biotech). Cy5-labeled sequencing products were
detected on line with an ALF express sequencer (Amersham Pharmacia Biotech).
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RESULTS |
In Vitro Analysis of the Upstream Extent of the Pea atp9
Promoter--
Initial studies of the mitochondrial pea atp9
promoter in a homologous in vitro transcription system had
confirmed the functional importance of the nonanucleotide between
nucleotides
7 and +2, which is conserved between several promoters.
In addition the analysis of clones containing 5'-deleted promoter
regions suggested that crucial sequence information for the function of
this promoter is located between nucleotide positions
25 and
8
relative to the 5' end of the nascent transcript (21). To identify the
functional requirements within this region, a series of deletion clones
was constructed and assayed in the in vitro transcription
system. While constructs with deletions upstream of positions
25,
21, and
16 contain full promoter function, the initiation activity is significantly reduced in construct
-12 and completely lost in
mutant
-7 (data not shown). For further resolution of the nucleotide
requirements in the comparatively AT-rich region between positions
16
and
7, the mitochondrial sequences were deleted in smaller steps
(Fig. 1, upper part). While
promoter function is not impaired in constructs
-16 and
-14,
activity is entirely lost in deletion clones
-10 and
-7. Partial
transcription initiation activity is again observed with clone
-12.
Independent reruns of these experiments, which were calibrated by the
addition of T7 RNA polymerase-generated transcripts to the in
vitro transcription assays, confirmed that the reduced amount of
run-off products originated from decreased intrinsic promoter activity
of this template and not from a selective loss of in vitro
generated RNAs during the assays (Fig. 1, lower part,
T7(161 Nt)). These functional analyses demonstrate that
sequences extending up to nucleotide position
14 are sufficient and
necessary to support transcription initiation at the pea
atp9 promoter in vitro.

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Fig. 1.
Definition of the upstream extent of the pea
atp9 promoter by deletion analysis. Sequences
upstream of the conserved nonanucleotide motif (gray box)
were successively deleted and replaced by vector sequences between
nucleotide positions 16 to 7 relative to the transcription
initiation site (upper part). The resulting clones ( -16
to -7) were linearized and tested in the pea in vitro
transcription system. The in vitro products were analyzed by
polyacrylamide gel electrophoresis (lower part). Designation
of the lanes corresponds to the respective template constructs. Size
variations between the lengths of DNA marker (lane M, sizes
given in nucleotides) and the 355 nucleotides in vitro
transcripts (indicated by an arrow on the right-hand
side) are due to the different migration velocities of DNA and RNA
molecules in the polyacrylamide gel electrophoresis. Transcripts
generated by T7 RNA polymerase (161 Nt(T7)) are added to
monitor the performance of individual in vitro assays.
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Sequences Extending to Nucleotide Position
14 Are Generally
Sufficient for Promoter Function in Vitro--
To gain more
information about the general significance of 5' sequences in other
dicot promoters, analogous deletion studies were carried out with the
soybean atp9 and the Oenothera rpl5 promoters,
respectively. In the assays with soybean constructs no functionally
required sequences are lost upon deletion upstream of position
-14,
but promoter activity is seriously reduced in clone
-12 (Fig.
2). In Oenothera rpl5 deletion
constructs no significant differences in transcription efficiencies
were found between
-17,
-14, and
-13. Promoter activity is,
however, completely abolished in deletions
-10 and
-7 in both
soybean atp9 and Oenothera rpl5 promoters. In
summary these results support the conclusion that the promoter portions
downstream of nucleotide position
14 are generally sufficient and
also necessary to drive transcription at mitochondrial promoters of
dicot plants. This region with a high content of adenines and
thymidines, which is already conspicuous in the sequence alignment of
several dicot promoters, has been accordingly designated AT-Box
(21).

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Fig. 2.
The AT-Box is indispensable for promoter
function in dicot plants. Deletions of mitochondrial sequences
between nucleotide positions 16 and 7 relative to the transcription
initiation sites of the soybean atp9 (A) and
Oenothera rpl5 (B) genes, respectively, show the
general significance of the AT-rich sequence element for promoter
function in dicot plant species. Clones, lanes, and transcripts are
marked analogously to Fig. 1.
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Transversion Mutagenesis of the AT-Box--
The high content in
adenosines and thymidines in the above described 5' regions between
nucleotides
14 and
8 of the mitochondrial promoters in dicot plants
suggests a function of these sequences in loosening or melting of the
DNA strands during the initiation process. To determine whether this
concentration of A and T nucleotides per se or whether
sequence specificity is important, adenosine and thymidine residues in
this particular region of the pea atp9 promoter were
transverted to the respective complementary nucleotides (Fig.
3, upper part). The melting
temperatures of the mutated and the wild type promoter regions were
calculated to differ by only 0.6 °C, and thus comparable initiation
efficiencies are expected if the function of this segment is confined
to access strand opening of the DNA. However, in vitro
transcription assays with the mutated and wild type templates revealed
that initiation is almost completely abolished by the transverted
nucleotides (Fig. 3, lower part). Semiquantitatively
repeated assays confirmed the significant reduction of the promoter
activity. The faint signal is only visible upon long exposures, which
indicates the residual promoter function to be less than 1% of the
wild type activity. This observation suggests that sequences rich in
adenosines and thymidines per se do not support promoter
function and that this promoter segment contributes to the
sequence-specific interaction(s) between the promoter and the
transcriptional apparatus in dicot plant mitochondria.

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Fig. 3.
Nucleotide identities within the AT-rich
region are essential for promoter function. Adenosine and
thymidine nucleotides in the AT-Box ( 14 to 8) of the pea
atp9 promoter were transverted to their complementary
identities (boxed lowercase letters). Designations of
the lanes correspond to those of the mutated (lane A/T TM)
and wild type (wt) templates. Run-off products (355 Nt) are highlighted by an arrow on the right-hand
side. The conserved nonanucleotide motif and the AT-Box are marked
by gray and hatched boxes, respectively.
Numbering of the sequence refers to the transcription start site
(+1).
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Insertions of 1, 3, 5, or 11 guanosine nucleotides between the AT-Box
and CNM also almost completely abolished promoter function. Since
promoter activity is not restored even when the distance between the
two parts corresponds to a complete helix turn, the AT-Box and CNM
appear to be interdependent in function (data not shown).
Nucleotides Downstream of the Transcription Start Site Contribute
to Promoter Function--
Analogous to the definition of the 5' border
of different dicot promoters, their 3' extent was investigated with the
pea atp9 transcription initiation site. Two 8-bp segments
ranging from +3 to +10 (LS-1) and from +11 to +18 (LS-2) were replaced
by 15- and 14-bp insertions of alien sequences, respectively. In
vitro transcription assays with both templates indicate that the
exchanged sequences are not important for promoter activity at least
in vitro (data not shown). To extend this analysis further
upstream into the conserved nonanucleotide motif, in three clones
segments between nucleotides
3 to +2 (TM-1), +3 to +7 (TM-2), and +8
to +12 (TM-3) were transverted to the respective complementary
sequences (Fig. 4A,
upper part). The correct transcripts of 355 nucleotides obtained with construct TM-3 confirmed that the region downstream of
position +8 does not participate in transcription initiation in
vitro. The ability to initiate transcription is however
completely lost in mutants TM-1 and TM-2 (Fig. 4A,
lower part). While the loss of promoter activity in TM-1 can
be explained by the disruption of the CNM, the inactivity of mutant
TM-2 contradicts the relatively normal promoter activity observed with
construct LS-1. To clarify these apparently inconsistent results, an
additional mutant was generated, in which nucleotides +5 to +7 were
transverted (Fig. 4B, TM-2', upper
part). While in vitro transcription assays with constructs LS-1 and TM-2 confirmed the previous results, mutant TM-2'
does retain promoter activity. In summary these results show that
sequences extending into transcribed regions up to nucleotide position
+4 are sufficient and at least partially required to drive plant
mitochondrial transcription in vitro.

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Fig. 4.
Sequences extending four nucleotides
downstream of the transcription initiation site are necessary but also
sufficient for promoter function in vitro.
A, segments of five nucleotides (boxed) are
transverted to the respective complementary nucleotides
(lowercase letters) in the 3' portion of the pea
atp9 promoter. B, construct TM-2', with sequences
between +5 and +7 substituted by complementary nucleotides, was
transcribed in vitro and compared with constructs TM-2 and
LS-1. Externally generated transcripts (161 Nt(T7)) are
added to detect variations during the purification of the run-off
products. Arrows indicate run-off products of correct sizes
(355 and 362 Nt, respectively). Lanes are
designated with respect to the templates used in the assays. Numbering
of the sequence, DNA size marker lengths and indication of essential
features of the promoter are identical to Fig. 3.
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18 Nucleotides Are Sufficient to Direct Transcription Initiation
Irrespective of Surrounding Sequences--
The above results of the
functional analysis of the 5' and 3' regions of the pea atp9
promoter imply that transcription should be initiated at an 18-bp
sequence segment (
14 to +4) irrespective of 5'- and 3'-flanking
regions. A double-stranded oligonucleotide (MP) representing such a
mitochondrial promoter was cloned into three different sequence
contexts and tested for transcription initiation (Fig.
5). The MP oligonucleotides integrated
into the SmaI and EcoRV restriction sites of the
pBluescript multiple cloning sites show the same full promoter activity
as the respective control template with this sequence in its wild type
context (pS and pE, Fig. 5, A and
B).

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Fig. 5.
An 18-nucleotide promoter sequence displays
full promoter function in vitro. Oligonucleotide
MP representing the wild type sequence of the atp9 promoter
(wt) between nucleotides 14 and +4 was cloned into
pBluescript vectors. A, construct pS containing
oligonucleotide MP in the SmaI site was linearized with
NaeI and transcribed in vitro. The run-off
transcripts of 372 nucleotides (pS) and 355 nucleotides
(wt) are indicated. No specific transcript is initiated at a
linearized pBluescript template without the promoter oligonucleotide
(MP) (C). Fragment sizes of a coelectrophoresed DNA marker
(M) are given at the left-hand side.
B, one (pE) and two MP oligonucleotides (pE
x2) were cloned into the EcoRV site of pBluescript.
Run-off transcripts of 390 and 408 nucleotides, respectively, indicate
correct transcription initiation at the inserted promoters. Marker
(M) and control reactions (lanes C and
wt) were identical to the ones in A.
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Two 18-mer promoter sequences inserted in tandem into the
EcoRV restriction site yield two transcription products
differing by 18 nucleotides in length (pEx2, Fig.
5B). No specific transcription product is observed with the
pBluescript vector alone clearly correlating transcription initiation
with the integrated MP oligonucleotide(s). Insertion of the MP
oligonucleotide into a SnaBI site within the pea
rps14/cob spacer region confers transcription
initiation to this region where no promoter is observed in
vivo and in vitro in the wild type sequence (data not
shown). These results conclusively demonstrate that the sequence from
14 to +4 represents a fully functional di- cot plant
mitochondrial promoter autonomous of adjacent sequences.
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DISCUSSION |
The Mitochondrial Promoter in Dicot Plants Is a Single Entity of 18 Nucleotides--
The functional analyses of several promoters by
deletions and transversions 5' and 3' to the transcription start
sites show that 18 nucleotides from
14 to +4 are essential and
sufficient for transcription in vitro. Insertion of the
18mer DNA segment confirms its fully competent promoter function
independent of 5'- and 3'-flanking sequences. This comprehensive
analysis thus identifies extension and structure of such a promoter.
To evaluate the experimental results, the respective regions of eleven
promoters from dicot plants were reinvestigated for conserved
structures (Fig. 6). Only transcription
initiation sites were included in this comparison, which have been
unambiguously identified as such by primer extension (in
vivo) and in vitro capping and/or in vitro
transcription analysis (in vitro) (12-14, 24).2
Most striking features within this 18-mer promoter sequence are the CNM
(
7 to +2) and the upstream AT-Box (
14 to
8). Although the
apparently noncanonic nucleotide identities separate these two motifs,
the in vitro analyses do not support individual
functions of these different promoter portions. The promoter sequence
rather appears to function as a single element, in which most
nucleotides are involved in sequence-specific interaction with the RNA
polymerase and/or additional transcription factor(s).

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Fig. 6.
Conserved structure of mitochondrial
promoters in dicot plants. Promoter sequences of 11 bona
fide promoters of dicot plants are compared between nucleotide
positions 14 and +4. The frequency with which each nucleotide
identity (given at the left margin) is found in any position
(top line) is given in percent. Predominant nucleotide
identities are given in the consensus sequence. Character sizes in the
consensus reflect the frequency with which the nucleotide identity is
found at the given position. Promoters compared in this analysis are
located upstream from the following genes: atp9, 18 S rRNA,
and rpl5 genes from pea; atp1, cox2,
rpl5, trnF, 18 S rRNA from Oenothera;
atp9 and rb/c from soybean; and the 18 S rRNA
gene of potato (12-14, 21, 24).2
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Requirement of the Primary Structure of the AT-Box for Promoter
Function--
Deletions in the 5' portion of three different promoters
from three dicot plant species consistently show that functional sequences maximally extend to position
14. Transcription initiation at the Oenothera rpl5 construct
-13 occurs with an
efficiency equal to clone
-14. This experimental result and the
observation that the adenosine at position
14 is less conserved
between different promoters (36%) suggest that this nucleotide
position may not be crucial for the activity of all of these promoters
(Figs. 1, 2, and 6).
Speculations that the AT-Box may only be necessary to facilitate
melting of the DNA strands are rendered highly unlikely by the almost
complete loss of activity in the transverted AT-Box mutant. This
sequence dependence is reflected in the high conservation at positions
13 (73% A),
12 (82% A),
11 (91% A), and
10 (73% T) within
this promoter segment. These observations strongly support this part of
the promoter to be involved in sequence-specific interaction with the
transcription apparatus, which does of course not exclude a parallel
positive influence of these AT-rich sequences on melting of the DNA strands.
The increased promoter activity in constructs
-14 relative to
-16, which is reproducibly observed in independent experiments with
both pea and soybean atp9 constructs, suggests that
sequences upstream of position
14 may somehow modulate promoter
strength. Thus substitution of nucleotide identities with negative
impact on promoter activity could result in a positive effect, a
phenomenon, which has to be investigated in detail by
site-directed mutagenesis at these particular nucleotides.
Adenosines in positions
12 to
10 have been observed to also
influence the activity of the maize atp1 promoter.
Substitution of the
12 A by a T or C considerably reduced the amount
of run-off transcripts (between 30 and 50%) of the wild type level
(18). These similarities between the 5' regions of the maize
atp1 and the dicot promoters investigated here may indicate
analogous promoter structures and recognition mechanisms. In the maize
cox3 promoter the respective segment was, however, found to
have only a minor role in transcription initiation in vitro
suggesting major differences of sequence requirements between
individual promoters in this plant species (19).
Nucleotides Downstream of the 5'-CRTA-3' Are Conserved and
Essential for the Function of Promoters in Dicot Plants--
Alignment
and sequence comparisons of transcription initiation sites determined
by in vitro capping studies identified the 5'-CRTA-3'
tetranucleotide to be conserved between monocot and dicot plant
species. Consequently this motif was assigned an important role in
promoter function (15, 16, 25). Various in vitro promoter
studies, however, showed that deviations are accepted in the first two
positions of this tetranucleotide in both plant subdivisions (18,
26).2 The small size of the motif and the relaxed sequence
constraints indicate that other nucleotides must be essential for
promoter function. In addition to the importance of the upstream
AT-Box, the nucleotides downstream of the 5'-CRTA-3' contribute
significantly to promoter function. This is highlighted by the complete
lack of initiation in the transversion mutant TM-1, in which the second half of the nonanucleotide sequence downstream of the 5'-CRTA-3' motif
is altered. The theoretical consensus deduced in the sequence comparison shows that this promoter segment contains three nucleotide positions at
2 (G), +1 (G), and +2 (A), which are 100% conserved in
all compared dicot promoters. Furthermore, two adenosine residues are
found at
3 and
1 with a frequency of 82%. This indicates that the
5'-CRTA-3' motif, although present in almost all mitochondrial promoters in seed plants, only represents a small fraction of the
nucleotides contributing more or less equally to the promoter function.
Purine Nucleotides Downstream of the Transcription Start Site
Contribute to Promoter Function--
The identification of the
nonanucleotide motif had already shown that the conserved promoter core
extends beyond the transcription start site. Nucleotide transversions
now show that the transcribed promoter portion actually extends even
further downstream, as far as nucleotide positions +3 and/or +4. Here
the actual requirements on nucleotide identity may be relaxed, since
not all substitutions downstream of position +2 interfere with
successful transcription initiation (Fig. 5). The differing results
obtained with constructs LS-1 and TM-2 may be due to the particular
nucleotide identities inserted at positions +3 and +4, these being a
guanosine at +3 in LS-1 and a thymidine residue in the respective
position in TM-2. Although guanosine or thymidine residues are found in
this position in some other promoters, at least one purine is found conserved at +3 or +4 in all wild type promoter sequences. Since TM-2
contains only pyrimidines in these positions, the loss of promoter
activity of this construct could be due to the total absence of purine
here. The precise requirements of nucleotide identities in these
positions have to be determined by site-directed mutagenesis.
Implications of the Refined Mitochondrial Promoter Structure in
Dicot Plant for the Interaction with the Transcription
Apparatus--
The analysis of the pea atp9 transcription
initiation site now shows that a mitochondrial promoter in a dicot
plant covers an 18-bp sequence, which corresponds to about two helix
turns. This promoter appears to act in its entirety as recognition site for components of the transcription apparatus. This may include one or
two proteins, which could, similar to transcription factors in
mammalian, yeast, and Xenopus mitochondria, direct a
promoter-specific attachment of the RNA polymerase (10, 11, 27, 28).
Two proteins that bind to this promoter have recently been enriched; however their direct participation in the transcription initiation process remains to be shown (29). All involved proteins must be in very
close proximity, since contact is made with the 18-bp promoter
sequence, which is rather short to accommodate attachment sites for
several different proteins, particularly considering that the RNA
polymerase itself already has a molecular mass of more than 100 kDa (7,
30).
The mode by which the transcriptional apparatus identifies a promoter
sequence is at present unclear, but can be refined with the results
presented here. The equal efficiencies with which transcription is
initiated at the tandemly repeated promoters exclude an access of the
proteins by scanning the DNA with a 5' to 3' processivity, since such
an access would result in a preferential transcription initiation at
the first promoter of this construct (Fig. 5). Thus, promoter
recognition in plant mitochondria rather appears to involve a homing
approach of one or more of the participating proteins.
The sequence requirements of mitochondrial promoters in dicot plants
defined here now provide a base toward the identification of the
components involved in transcription initiation. Furthermore, this
refined promoter sequence will allow rapid identification of other
mitochondrial promoters in this plant subdivision, e.g. in
the complete sequence of the Arabidopsis mitochondrial
genome (22, 31).