Plant Physiol. (1998) 116: 519-527
Characterization of DNA-Binding Proteins from Pea
Mitochondria1
Frank Hatzack2,
Saskia Dombrowski,
Axel Brennicke, and
Stefan Binder*
Allgemeine Botanik, Universität Ulm, Albert-Einstein-Allee
11, D-89069 Ulm, Germany
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ABSTRACT |
We
studied transcription initiation in the mitochondria of higher plants,
with particular respect to promoter structures. Conserved elements of
these promoters have been successfully identified by in vitro
transcription systems in different species, whereas the involved
protein components are still unknown. Proteins binding to
double-stranded oligonucleotides representing different parts of the
pea (Pisum sativum) mitochondrial atp9
were analyzed by denaturation-renaturation chromatography and
mobility-shift experiments. Two DNA-protein complexes were detected,
which appeared to be sequence specific in competition experiments.
Purification by hydroxyapatite, phosphocellulose, and reversed-phase
high-pressure liquid chromatography separated two polypeptides with
apparent molecular masses of 32 and 44 kD. Both proteins bound to
conserved structures of the pea atp9 and the
heterologous Oenothera berteriana atp1
promoters and to sequences just upstream. Possible functions of these
proteins in mitochondrial promoter recognition are discussed.
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INTRODUCTION |
In many genetic systems the level of expression of a gene is
controlled by modulating the efficiency of transcription through changing the concentrations and activities of transcription initiation factors.
Proteins involved in transcription initiation in mitochondria have been
purified and characterized from yeast (Saccharomyces cerevisiae) and animals (Tracy and Stern, 1995
). In yeast two nuclear-encoded proteins are required for the initiation of
transcription on a conserved nonanucleotide promoter, a core RNA
polymerase of 145 kD (sc-mtRNAP), and a 43-kD specificity factor
(sc-mtTFB; Kelly et al., 1986; Schinkel et al., 1987; Lisowsky and
Michaelis, 1988; Shadel and Clayton, 1993). Whereas the RNA polymerase
shows similarities to bacteriophage RNA polymerases (Masters et al., 1987), sc-mtTFB resembles bacterial 
factors (Jang and Jaehning, 1991). Transcriptional initiation is preceded by promoter-specific DNA
binding of a complex of both proteins (Schinkel et al., 1988; Magus et
al., 1994). A third factor potentially involved in the initiation
process is sc-mtTFA, a 19-kD protein containing two HMG-like
DNA-binding domains. sc-mtTFA has a moderately stimulating effect on
transcriptional initiation in vitro, most likely due to its primary
function of compacting and organizing yeast mtDNA (Diffley and
Stillman, 1991; Fisher et al., 1991, 1992; Parisi et al., 1993).
In xl mitochondrial transcription is similarly initiated with the
approximately 140-kD RNA polymerase xl-mtRNAP, requiring the
dissociable factor xl-mtTFB, which is approximately 40 kD (Bogenhagen
and Insdorf, 1988). Both proteins have been isolated in high purity,
but their respective genes have not yet been cloned. The 30-kD
xl-mtTFA, a protein with two HMG boxes, binds preferentially to the
control region adjacent to the major promoters of xl-mtDNA and shows
pronounced stimulation of transcription in vitro, similar to the
homologous protein in yeast (Antoshechkin and Bogenhagen, 1995).
Mitochondrial transcription factors of the mtTFA family have also been
purified and cloned from humans (h-mtTFA, 25 kD; Fisher and Clayton,
1988; Fisher et al., 1991; Parisi and Clayton, 1991) and mice (m-mtTFA,
23.5 kD; Larsson et al., 1996). Both proteins bind specifically to
regions located upstream of the respective mitochondrial promoters and
have a strong enhancing effect on the initiation of transcription
(Larsson et al., 1996; Fisher et al., 1987, 1989). The h-mtRNA
polymerase has recently been identified in mammalia as a phage-like,
single-subunit enzyme that is similar to the respective yeast protein
(Tiranti et al., 1997).
Investigation of the transcriptional initiation process in plant
mitochondria has so far been focused on structural promoter studies by
in vitro transcription analysis. Such in vitro systems have been
established for wheat (Triticum aestivum; Hanic-Joyce and
Gray, 1991), maize (Zea mays; Rapp and Stern, 1992), and pea (Pisum sativum; Binder et al., 1995) mitochondria. The
architecture of the atp1 promoter in maize was analyzed
by deletion analysis, linker-scanning analysis, and site-directed
mutagenesis. These investigations identified a CRTA motif, conserved
among monocot promoters, to represent the core promoter with an A-rich
upstream domain enhancing initiation efficiency (Rapp and Stern, 1992; Rapp et al., 1993). A similar, although more extended nonanucleotide motif, 5
-(
7)CRTAAGAGA(+2)-3, was found to surround transcription initiation sites in many promoters from dicot plants. Transcriptional studies with a pea mitochondrial lysate showed that the conserved nonanucleotide motif is recognized in homologous and heterologous in
vitro transcription reactions, suggesting common transcriptional features among dicot plants. A 5 deletional analysis of the
atp9 promoter from pea mitochondria determined a minimal
upstream region required for efficient in vitro initiation. The
functional promoter structure extends up to nucleotide position 25
and consists of a nonanucleotide motif and, as in monocot plants, an
upstream A(+T)-rich region (Binder et al., 1995).
The pea atp9 promoter structure now provides a template to
search for DNA-binding proteins, which specifically recognize this region and thus could be involved in the transcription initiation process. As a purification strategy for such proteins we used the
method of Fisher et al. (1991), a sequence of purification steps under
denaturing and native conditions. Since this approach was successfully
used for the purification of mtTFA proteins from the evolutionary
distant organisms S. cerevisiae and humans (cell culture),
we reasoned that this method might also represent a suitable strategy
with which to purify a similar factor from plant mitochondria.
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MATERIALS AND METHODS |
Preparation of ds-Oligonucleotides
ds-Oligonucleotides were prepared by annealing equi-molar
amounts of two complementary single-stranded oligonucleotides in 50 mm Tris-HCl, pH 7.6, 10 mm
MgCl2, 5 mm DTT, 1 mm
spermidine, and 1 mm EDTA. Annealing reactions (50 µL)
were incubated at 90°C for 2 min and slowly cooled to room
temperature overnight. Oligonucleotides were obtained by annealing the
following single-stranded oligonucleotides with the corresponding
antisense oligonucleotides (not shown): P1 (PA-79:
5-GAACTGCTTGCTTATGTGAGGTTCTTT-3), P2 (PA-52:
5-CCTCTCGCTTGTTCATCTTGTTTTGAG-3), P3 (PA25:
5-TACTCGACGAAATAATAGCATAAGAGA-3), P4 (PA+3:
5-AGATATTGGACATTGAGTCCACTTCG-3), P5 (PA+30:
5-ATATCACACCTATTTGAGTCGGGAGTT-3), O1 (OA-79:
5-GAGCACATCGAAATTTCCAATCCGGTT-3), O2 (OA-52:
5-CCAAGCCAGGTAAGCAAGTTCCT-TTTC-3), O3 (OA-25:
5-TAAAGAAAGTTGATAAATCAT-AAGAGA-3), O4
(OA+3: 5-AGCAAAGTCCCTAGTC-AAAGGTGGTTG-3), O5
(OA+30: 5-GGAAGTAGTACG-CCCGGTTCACAGGTT-3), PtrnF
(PIII: 5-CTCTTGTC-TTCCGTCTTTTG-3), and Pnad5 (Exc+:
5-TACCTAAAC-CAATCATCATATCGAC-3).
Radioactive Labeling of ds-Oligonucleotides
Thirteen picomoles of ds-oligonucleotides was 5-labeled by T4
polynucleotide kinase (Boehringer Mannheim) with 70 µCi of [
-32P]ATP (3000 Ci/mmol). Labeling reactions
were incubated for 45 min at 37°C. Microspin S200 columns (Pharmacia)
were used according to the manufacturer's instructions to remove
unincorporated [-32P]ATP.
ds-Oligonucleotide concentration and total radioactivity were
determined by photometric measurement and scintillation counting, respectively. The specific activities of the probes varied between 800 and 2000 cpm/fmol.
Isolation of Pea Mitochondria
Pea (Pisum sativum L., var Lancet) seedlings were grown
in the dark for 7 d. Mitochondria were isolated and purified as
described previously (Binder and Brennicke, 1993).
Denaturation-Renaturation Chromatography of Mitochondrial Proteins
Purified pea mitochondria (approximately 2 g) were carefully
resuspended in 30 mL of ice-cold buffer W (100 mm
NaPO4, pH 6.8, 250 mm Suc, 15%
glycerol, 1 mm DTT, and 1 mm PMSF) and
centrifuged for 15 min at 27,000g. Mitochondria were then
resuspended in 50 mL of boiling lysis buffer L (100 mm
NaPO4, pH 6.8, 2% SDS, 20 mm DTT,
and 1 mm PMSF). The mixture (approximately 40 mg of total protein) was boiled for 5 min until the lysate had cleared and was then
diluted 10-fold with buffer D (100 mm
NaPO4, pH 6.8, 1 mm DTT, and 1 mm PMSF).
Hydroxyapatite chromatography (Bio-Rad) was performed at room
temperature on a 70-mL column equilibrated in buffer D adjusted to
0.1% SDS. The column was loaded with the diluted mitochondrial lysate
and extensively washed with buffer D containing 0.1% SDS. Elution was
performed with a linear gradient of 100 to 500 mm NaPO4, pH 6.8, in 0.1% SDS, 1 mm
DTT, and 1 mm PMSF. Fifty fractions of 4 mL each were
collected and assayed for DNA-binding activity by mobility-shift
experiments. Hydroxyapatite fractions with DNA-binding activity were
pooled, renatured by adding 2 volumes of renaturation buffer RII (20 mm Tris-HCl, pH 8.0, 0.2 mm EDTA, 15%
glycerol, 4% Triton X-100, 2 mm DTT, and 0.5 mm PMSF), and incubated on ice for 20 min.
Chromatography on phosphocellulose (Sigma) was carried out at 4°C.
Renatured hydroxyapatite fractions were loaded onto a 20-mL column
equilibrated in phosphocellulose buffer (10 mm Tris-HCl, pH
8.0, 0.1 mm EDTA, 7.5% glycerol, 0.1% Triton X-100, 1 mm DTT, and 0.75 mm PMSF) containing 100 mm NaCl. Proteins were eluted with a linear gradient of 0.1 to 1.0 m NaCl in phosphocellulose buffer. Fifty 5-mL
fractions were collected and assayed for DNA-binding activity.
Mobility-Shift Assays
Aliquots of hydroxyapatite fractions were renatured by adding 4 volumes of renaturation buffer RI (9 mm Tris-HCl, pH 8.0, 45 mm KCl, 0.9 mm EDTA, 44.5% glycerol, 2%
Triton X-100, and 1 mm DTT) and incubating on ice for 20 min. Two microliters of the renatured protein fractions was mixed with
5 µL of hydroxyapatite shift mixture (20 mm Tris-HCl, pH
8.0, 20 mm MgCl2, 200 µg/mL BSA,
and 100 mg/mL ds-poly[d{I-C}]), 1 µL of radiolabeled probe (approximately 15,000 cpm), and 2 µL of double-distilled water. In
the phosphocellulose fractions, 2-µL aliquots were added to 7 µL of
phosphocellulose shift mixture (14.3 mm Tris-HCl, pH 8.0, 14.3 mm MgCl2, 35.7% glycerol, 140 mg/mL BSA, 140 µg/mL ds-poly[d{I-C}]), and 1 µL of
radiolabeled probe (approximately 15,000 cpm). Binding reactions were
incubated at 25°C for 20 min, supplemented with 1 µL of loading
solution (200 mm Tris-HCl, pH 8.0, 0.1% [w/v] bromphenol
blue, 0.1% [w/v] xylenecyanole, and 50% glycerol) and electrophoresed on native 4% polyacrylamide gels
(acrylamide:bisacrylamide, 30:1) in 10 mm Tris-HCl, pH 8.0, 5 mm sodium acetate, and 1.5 mm EDTA.
rpHPLC
rpHPLC was performed on a C4 column (Bioselect, Eurosil, Knauer,
Berlin, Germany; 4.6 × 250 mm, medium pore size, 300 Å). Fifteen
milliliters of pooled phosphocellulose fractions was loaded onto the
column equilibrated in an aqueous solution containing 5% acetonitrile
and 0.1% trifluoroacetic acid. Proteins were eluted by a linear
gradient of 5 to 80% acetonitrile in 0.1% aqueous trifluoroacetic
acid. One-hundred 1-mL fractions were collected. Fractions were
lyophilized and the pellets were resuspended in 10 µL of buffer D
adjusted to 100 mm NaPO4. After
incubation for 30 min at room temperature, 2-µL aliquots were
renatured as described for hydroxyapatite fractions and then
tested for DNA-binding activity in mobility-shift experiments.
Recovery and Renaturation of Proteins after Purification by
SDS-PAGE
SDS-polyacrylamide gel slices were covered with 350 µL of
elution buffer (50 mm Tris-HCl, pH 8.0, 0.1 mm
EDTA, 150 mm NaCl, 10% glycerol, and 1 mm DTT)
and crushed with a glass pestle until fine gel slurries were obtained.
After an overnight incubation at 4°C, the slurries were pipetted onto
ultrafiltration tubes (Microcon 10, Amicon, Beverly, MA) equipped with
prefilters (pore size, 0.2 µm). Samples were centrifuged at
8500g for 60 min to separate the eluted proteins from
polyacrylamide debris and to concentrate the proteins. The final volume
of the concentrated proteins was 5 to 15 µL. Aliquots with a volume
of 0.5 µL were renatured by the addition of 2 µL of renaturation
buffer RI and tested for DNA-binding activity. The remaining proteins
were supplemented with 10 µL of sample buffer (125 mm
Tris-HCl, pH 6.8, 2% SDS, 17% glycerol, 0.01% bromphenol blue
[w/v], and 5% [v/v] 
-mercapto-ethanol), resolved alongside
a molecular weight marker (Amersham) on analytical 12.5%
SDS-polyacrylamide gels (Laemmli, 1970), and detected by silver
staining.
Determination of Relative Binding Activity
Mobility-shift reactions were performed with renatured rpHPLC
fractions containing one of the two DNA-binding proteins (fraction no.
49, a 32-kD protein, or fraction no. 51, a 44-kD protein) and different
ds-oligonucleotides (P1-P5 or O1-O5). The total radioactivity in each
binding reaction was adjusted to 15,000 cpm. DNA-protein complexes were
detected by autoradiography with preflashed radiographic films
(Hyperfilm-MP, Amersham), and absolute signal intensities were
determined by densitometric analysis using a scanner (model ED-8000,
Epson, Torrance, CA) and imaging software (ImageQuant 2.1, Molecular
Dynamics, Sunnyvale, CA). Absolute signal intensities were corrected by
subtracting local background intensities and then multiplying with
normalizing factors to compensate for differences resulting from the
different specific activities of the ds-oligonucleotides in each
experiment. Relative binding was compared by setting the highest
normalized signal intensity determined in one experiment to 100%.
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RESULTS |
Purification of DNA-Binding Proteins from Pea Mitochondria by
Denaturation-Renaturation Chromatography
Proteins from pea mitochondria binding to ds-oligonucleotides
representing a mitochondrial promoter were purified with the denaturation-renaturation chromatography procedure developed by Fisher
et al. (1991; Fig. 1A).
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| Figure 1.
Purification of DNA-binding proteins from pea
mitochondria. A, Purification scheme. Pea mitochondria were denatured
by boiling in an SDS-containing buffer. The SDS-lysate was loaded onto
a hydroxyapatite column and eluted with a linear NaPO4
gradient (0.1-0.5 m). Aliquots of the eluted fractions
were renatured and tested for DNA-binding activity in mobility-shift
experiments (B). Fractions with binding activity (nos. 22-28) were
pooled, renatured, and loaded onto a phosphocellulose column. The
column was eluted with a linear NaCl gradient (0.1-1 m),
and fractions were assayed for DNA-binding activity by mobility-shift
analysis (C). The approximate NaPO4 and NaCl concentrations
of fractions with the highest binding activities are indicated at the
upper margins. Arrows in the right margins indicate the positions of the unbound ds-oligonucleotide P3 and of the DNA-protein complexes C1
and C2. Fraction numbers are given at the bottom in B and C.
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DNA-binding activity of eluted and renatured column fractions was
assayed by mobility-shift analysis with a labeled ds-oligonucleotide (P3, 27 bp) covering the promoter core structure of the pea
atp9 gene from 25 to +2 relative to the first transcribed
nucleotide. This sequence was sufficient in vitro to support
transcription initiation and did not require any further upstream or
downstream sequences (Binder et al., 1995; S. Dombrowski and S. Binder,
unpublished results). Two distinct DNA protein complexes, C1 and C2,
were identified in fractions eluting at approximately 320 mm NaPO4 from the hydroxyapatite
column (Fig. 1B). Fractions containing binding activity were pooled,
renatured, and chromatographed on a phosphocellulose column. Pronounced
C1 and C2 formation was observed in fractions eluting at approximately
330 mm NaCl (Fig. 1C).
Analysis of phosphocellulose fractions by SDS-PAGE did not allow a
clear correlation between individual protein bands and the binding
activities (data not shown), and additional purification steps were
thus required to identify the DNA-binding proteins. Prior to further
enrichment procedures, we investigated to what extent the observed
complex formation is specific for the atp9 promoter
sequence.
Analysis of DNA-Binding Specificity
Competition experiments with different ds-oligonucleo-tides
offered a convenient approach for determining how specific proteins in
complexes C1 and C2 recognize the atp9 promoter sequence.
Binding reactions were performed with the 330 mm NaCl
fraction from the phosphocellulose column (fraction no. 14) and labeled
oligonucleotide P3, which covers the entire promoter (Fig.
2). A 10-fold molar excess of unlabeled
P3 was found to be sufficient for almost complete suppression of the C1
and C2 signals (Fig. 2; lane 3). In contrast, the heterologous
competitors PtrnF (20 bp) and Pnad5 (22 bp) only slightly reduced
complex formation when present in 20- or even 40-fold molar excess
(Fig. 2; lanes 4-7). The two competitor sequences are parts of
mitochondrial genes and have no similarity to the promoter consensus.
These results suggest that the DNA-binding proteins in C1 and C2 have,
to some extent, sequence-specific binding properties.
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| Figure 2.
Competition experiments. Aliquots of the 330 mm NaCl phosphocellulose fraction were incubated with the
labeled ds-oligonucleotide P3 in the presence of unlabeled P3 (lane 2)
and the heterologous competitors PtrnF (20 bp, lanes 4 and 5) and Pnad5
(22 bp, lanes 6 and 7). The molar excesses in which the competing DNA
molecules were added over P3 are indicated in the second line at the
top. Binding reactions in the absence of mitochondrial proteins (lane 1) and in the absence of competitor (lane 2) served as negative and
positive controls, respectively. A homologous competition experiment
was performed to determine the molar excess of unlabeled P3 required
for an almost complete suppression of the C1 and C2 signals (lane 3).
Arrows at left indicate the positions of the DNA-protein complexes C1
and C2 and of the unbound ds-oligonucleotide P3.
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Separation of the Two DNA-Binding Activities by rpHPLC
The denaturation-renaturation chromatography of pea mitochondrial
proteins showed that the DNA-binding activity of the proteins in C1 and
C2 can be recovered after denaturation in SDS. This observation allows
the application of rpHPLC as a next purification step. rpHPLC has a
high protein-separation capability but has to be carried out under
partially denaturing conditions, since organic solvents are used for
elution. Therefore, proteins purified by rpHPLC must be renatured
before activity assays can be conducted.
Phosphocellulose fractions with DNA-binding activity (Fig. 1C, fraction
nos. 6-20) were pooled and chromatographed on an rpHPLC C4 column. The
two DNA-binding activities were efficiently separated by the rpHPLC
(Fig. 3A). The highest C1 formation was
observed with fraction no. 51, which was collected at 49.5%
acetonitrile, whereas the peak of C2-binding activity was detected
in fraction no. 49, which was collected at 47.8% acetonitrile.
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| Figure 3.
Separation of DNA-binding proteins by rpHPLC. A,
Phosphocellulose fractions with binding activities (Fig. 1C, fraction
nos. 6-20) were pooled and chromatographed on an rpHPLC C4 column. Fractions eluted with a linear acetonitrile gradient (5-80%) were tested after renaturation for DNA-binding activity by mobility-shift analysis using ds-oligonucleotide P3. Arrows in the right margin indicate the positions of the DNA-protein complexes C1 and C2 and of
the unbound ds-oligonucleotide P3. Fraction numbers are given at the
bottom. B, SDS-PAGE analysis of rpHPLC fraction nos. 49 and 51. Proteins were resolved on 12.5% SDS-polyacrylamide gels and visualized
by silver staining. Molecular masses of marker proteins are given in
kilodaltons.
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The separation of DNA-binding activities confirms that C1 and C2 are
formed by at least two different proteins rather than by dimeric and
monomeric complexes of the same protein. SDS-PAGE analysis showed that
the protein compositions in the fractions with peak binding activities
are still too complex to allow assignment of the DNA-binding proteins
(Fig. 3B).
Size Determination of the Two DNA-Binding Proteins
The apparent molecular masses of the proteins in complexes C1 and
C2 were estimated by gel electrophoresis. rpHPLC fractions with peak
binding activities (Fig. 3A, fractions 49 and 51) were resolved by
electrophoresis on preparative SDS-polyacrylamide gels, which were
subsequently cut into 3-mm slices. Proteins from individual gel slices
were recovered by elution, and aliquots were tested for DNA-binding
activity after renaturation (Fig. 4, A
and C). The remaining proteins were again loaded onto analytical SDS-polyacrylamide gels (Fig. 4, B and D) and co-electrophoresed with a
molecular size marker (lanes M) and with aliquots of the original
rpHPLC fractions, which served as reference samples (lanes R). Apparent
sizes of proteins with DNA-binding activity could thus be estimated by
comparing protein bands present on the silver-stained gels with shift
signals in the corresponding lanes of the mobility-shift assays.
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| Figure 4.
Size determination of the purified proteins by
gel-dissection experiments. rpHPLC fraction nos. 49 and 51 were
electrophoresed on two separate, preparative 12.5%
SDS-polyacryl-amide gels (not shown). Each gel was cut into 10 slices, as indicated by the numbered boxes at the left of B and at the
right of D. A and C, Proteins were eluted from these gel slices and
aliquots of the eluted proteins were tested for DNA binding in
mobility-shift assays. Arrows indicate the signals of DNA-protein
complexes C1 and C2. Numbers of the polyacrylamide slices are given at
the bottom. B and D, The remaining proteins were electrophoresed on
analytical 12.5% SDS-polyacrylamide gels alongside an aliquot of
rpHPLC fraction no. 51 or no. 49 (lanes R in B and D). Proteins were
detected by silver staining. Numbers at the top correspond to the gel
slice numbers. Molecular masses of marker proteins are given in
kilodaltons (lanes M).
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Analysis of rpHPLC fraction no. 51 (Fig. 3A) identified a DNA-binding
protein with an apparent molecular mass of approximately 44 kD (Fig. 4,
A and B, lanes 8). A polypeptide migrating at 50 kD is present in all
lanes in which eluted proteins were resolved in the analytical protein
gel (Fig. 4B, lanes 1-10). This protein is absent from both the
reference and marker lanes (lanes R and M) and is therefore very likely
a contaminant that was introduced during elution or renaturation. Other
more diffuse bands in lane 8 are most likely degradation products of
the 44-kD protein, since they are too small to represent intact
proteins recovered from gel slice no. 8.
The analysis of rpHPLC fraction no. 49 (Fig. 3A) identified a
DNA-binding activity from gel slice nos. 3 to 5, containing proteins
with apparent molecular masses of 30 to 32 kD and forming complex C2
with P3 (Fig. 4, C and D, lanes 5). Signal structures in the
autoradiogram of the mobility-shift gel (Fig. 4C, lanes 3 and 4)
possibly identify more than one complex-forming protein with similar
sizes in the range of 30 to 32 kD (Fig. 4D). The resolution of complex
C2 achieved in the mobility-shift analyses of different mitochondrial
fractions did not show whether one or more complexes was formed by
different proteins in this size range (Figs. 1, B and C, and 4C). Since
there was at least one protein participating, we consider complex C2 as
being derived from one protein of 32 kD, without precluding the
involvement of others. Further specific investigation will be required
to resolve the question of additional DNA-binding polypeptides of this
size range.
DNA-Binding Specificity of the Two Proteins to Different Parts of
Two Mitochondrial-Promoter Regions
The competition experiments suggested that the detected proteins
from pea mitochondria show sequence-specific binding to the ds-oligonucleotides, representing a plant mitochondrial promoter region
(Fig. 2). To gather more experimental information about these proteins
and their potential for recognizing the promoter sequences, we
investigated binding to different parts of the pea atp9 and
the heterologous Oenothera berteriana atp1 promoter
regions and their sequence vicinities. The latter promoter was selected because it has been recognized and correctly activated in an in vitro
transcription assay in pea (Binder et al., 1995).
Two series of ds-oligonucleotides, P1 to P5 and O1 to O5, represent
27-bp sections flanking the respective transcriptional start sites
(Fig. 5). Binding reactions of the
labeled ds-oligonucleotides to the rpHPLC-purified proteins were
analyzed on 4% native polyacrylamide gels, and complex formation was
compared by densitometric evaluation of mobility-shift experiments. Two
independent sets of experiments (Fig. 6)
reveal the strongest shift signals and thus preferential binding of
both proteins, with ds-oligonucleotides representing the core promoter
(P3/O3) and the respective upstream oligonucleotides (P2/O2). Minor
binding is observed with P1 and O1 (covering regions further upstream),
and almost no binding to the downstream ds-oligonucleotides (P4/O4 and
P5/O5) is observed. This study suggests that the identified proteins
have similar binding properties, binding preferentially to DNA regions
containing the conserved promoter elements (P3/O3) and the
nonconserved, upstream sequences from both plant species (P2/O2).
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| Figure 5.
Schematic representation of ds-oligonucleotides
used in the investigation of protein binding in different sections of
two promoter regions. Two series of ds-oligonucleotides were used to
investigate protein binding in different sections of the pea atp9 (P1-P5) and the O. berteriana atp1
(O1-O5) promoter regions. Each ds-oligonucleotide represents a
different section of one promoter region and was used as the DNA
substrate in mobility-shift experiments (Fig. 6). The contiguous
sequences of the two ds-oligonucleotide arrays correspond to the
complete sequences of the atp9 and atp1 promoter regions from nucleotide position 79 to +56, respectively. Both promoter regions contain conserved nonanucleotide motifs (CNM) and
AT-rich regions (AT-box). Numbering of the nucleotide positions refers
to the transcriptional starting point (nucleotide position +1),
represented by a bent arrow.
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| Figure 6.
DNA-binding specificity in different sections of
the pea atp9 and the O. berteriana
atp1 promoter regions. A and C, Autoradiograms of
mobility-shift experiments. rpHPLC fractions containing either the
32-kD protein (fraction no. 49) or the 44-kD protein (fraction no. 51)
were used to assay C2 and C1 formation. Complexes are indicated by
arrows. The ds-oligonucleotides used in the individual reactions are
given at the top. Binding reactions were carried out in the presence
(+) or absence () of mitochondrial proteins. B and D, Quantitative
evaluation of two sets of mobility-shift experiments (autoradiograms in
A and C representing one set). Relative binding affinities of the 44- and 32-kD proteins to the individual ds-oligonucleotides were
calculated from normalized C1 and C2 signal intensities (see
``Materials and Methods''). Gray and black bars represent the
relative binding affinities derived from the first and the second data
sets, respectively.
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DISCUSSION |
DNA-Binding Proteins from Pea Mitochondria
The denaturation-renaturation method developed by Fisher et al.
(1991) and rpHPLC were used to purify proteins that preferentially bind
to mitochondrial promoter sequences from higher plants. This approach
identified 32- and 44-kD proteins from pea mitochondria. The 32-kD
proteins behave as distinct proteins in the SDS-PAGE analysis, whereas
their ds-oligonucleotide complexes cannot be clearly distinguished by
their gel migration; therefore they may represent variant protein
forms. The DNA-binding activities of these proteins are recovered after
denaturation and elute at similar conditions during chromatography on
hydroxyapatite, phosphocellulose, and rpHPLC (Figs. 1 and 3).
Comparison of the Pea Proteins with mtDNA-Binding Proteins from
Other Organisms
The denaturation-renaturation purification procedure used here to
enrich pea mitochondrial proteins was originally developed to purify
mtTFA proteins from yeast and animal mitochondria. It therefore appears
reasonable to compare characteristics of the proteins identified in pea
with those of sc- and h-mtTFA for similarities and differences in
biochemical properties.
The first similarity is their survival through the harsh denaturing
treatment and their regaining DNA-binding activity through renaturation
in the presence of Triton X-100. Furthermore, these two pea
mitochondrial proteins co-elute at approximately 320 mm NaPO4 from the hydroxyapatite column, comparable
to the NaPO4 concentrations required to elute
sc-mtTFA (approximately 330 mm) and h-mtTFA (approximately
300 mm) from this matrix. However, in phosphocellulose
chromatography, the pea proteins elute at approximately 330 mm NaCl, whereas significantly higher salt concentrations are required to recover h-mtTFA (580 mm) and sc-mtTFA (700 mm NaCl; Fisher et al., 1991). No DNA-binding activity is
detected with pea mitochondrial proteins mobilized at comparably high
NaCl concentrations. The pea 32- and 44-kD proteins thus show
purification characteristics quite different from those of the sc-mtTFA
and h-mtTFA polypeptides.
The 44-kD protein has a molecular mass similar to xl-mtTFB proteins
(approximately 40 kD; Bogenhagen and Insdorf, 1988; Antoshechkin and
Bogenhagen, 1995) and sc-mtTFB (43 kD; Schinkel et al., 1987). Although
the sc-mtTFB activity can also be recovered after denaturing SDS-PAGE
(Schinkel et al., 1987), it seems unlikely that the 44-kD pea
polypeptide represents a plant mtTFB homolog. In contrast to mtTFB,
which binds to promoters only in a binary complex with the respective
mitochondrial RNA polymerase, the 44-kD protein from pea shows
significant binding to promoter regions, and binding does not depend on
the presence of other proteins (such as the mtRNA polymerase, which is
more than 100 kD; Figs. 2 and 6).
The molecular masses of the identified pea proteins also suggest that
neither of them resembles a mitochondrial RNA polymerase, which is 112 kD in Chenopodium album (Weihe et al., 1997) and is most
likely of a similar size in mitochondria of plants in general,
including pea (Cermakian et al., 1996).
Recognition of Protein-Binding Sites in Plant Mitochondrial
Promoter Regions
The binding studies with the two series of ds-oligonucleotides
(Fig. 6) revealed that the formation of the respective DNA-protein complexes is a nonrandom, sequence-dependent process. The strongest protein binding was observed at two sites upstream of the transcription start sites in both the pea atp9 and the O. berteriana promoter regions. The proximal, preferred
protein-binding sites contain the two important sequence elements
identified in plant mitochondrial promoters. The conserved
nonanucleotide motif and the AT-box are covered by the
ds-oligonucleotides P3 and O3 (Fig. 5). Consequently, one or both
elements may be recognized by proteins binding immediately upstream of
the respective transcription start site. However, the more distal,
second protein-binding sites contained in P2 and O2 do not share any
extended sequence similarities, either with each other or with P3 and
O3. Therefore, sequence elements important for this second binding site
cannot easily be correlated with a conserved consensus sequence.
In mitochondrial light-strand promoters of human and mouse, h-mtTFA and
m-mtTFA specifically recognize such "hidden" elements. Here,
binding sites were identified by DNase I footprinting and further
analyzed by methylation interference assays. The DNase I footprints of
both proteins extend approximately between nucleotides 10 and 40
relative to the respective transcription start site. Although these two
upstream regions show only very limited sequence similarities, several
of the directly contacted nucleotides were found conserved between the
two species. Heterologous footprint experiments and in vitro
transcription reactions revealed that these cryptic, conserved features
are sufficient for specific mtTFA binding and transcriptional
stimulation (Fisher et al., 1987, 1989).
In plant mitochondrial promoters, similar investigations are required
to map binding sites more precisely and to identify critical
protein-nucleotide contacts. Such studies will reveal to what extent
the conserved nonanucleotide motif and/or the AT-box play a role in the
specific binding of the 32- and 44-kD proteins. The 5 deletional
analysis of the pea atp9 promoter suggests that engagement
of the protein-binding site in the conserved promoter region, the
P3-section, is, at least in vitro, sufficient for the effective
initiation of transcription (Binder et al., 1995). Therefore, protein
binding to additional upstream site(s) in the P2- or even in the
P1-section may have a more enhancing effect on the transcription
initiation process.
Are the Identified Pea Mitochondrial Proteins Connected with the
Initiation of Transcription in Plant Mitochondria?
The promoter-binding properties of the 32- and the 44-kD proteins
tentatively support a preferential connection of these polypeptides to
the transcription initiation process in pea mitochondria.
In addition, enhanced transcription initiation activity is observed
upon the addition of a fraction containing the two promoter-binding proteins to an in vitro transcription reaction (data not shown). However, this stimulating mitochondrial fraction (330 mm
NaCl phosphocellulose fraction; fraction 14 in Fig. 1) is far too
complex and has too many other proteins to allow a connection with the 32- and 44-kD proteins. Enhancing effects were observed in analogous in
vitro transcription experiments when h-mtTFA-containing protein fractions were incubated with partially purified human mtRNA polymerase (Fisher and Clayton, 1988) or when xl-mtTFA was added to mixtures of
xl-mtTFB and xl-mtRNAP (Bogenhagen and Insdorf, 1988).
The increased transcript amount could alternatively be explained by an
improved RNA stability. Preliminary investigations showed that the slow
degradation rate of T7 RNA polymerase transcripts of the pea
atp9 region in mitochondrial lysate preparations used for in
vitro transcription is not influenced by the addition of the 330 mm NaCl phosphocellulose fraction (data not shown). Future work should analyze the observed transcriptional effect in more detail,
which should elucidate the putative stimulatory function of each
DNA-binding protein separately.
| |
FOOTNOTES |
1
This work was supported by the Deutsche
Forschungsgemeinschaft, a Landesforschungsschwerpunkt
Baden-Württemberg, the Human Frontiers Science Program, and the
Fonds der Chemischen Industrie.
2
Present address: Plant Genetics, Environmental
Sciences and Technology Department, Risø National Laboratory, DK-4000
Roskilde, Denmark.
*
Corresponding author; e-mail stefan.binder{at}biologie.uni-ulm.de; fax
49-731-502-2626.
Received August 15, 1997;
accepted October 6, 1997.
| |
ABBREVIATIONS |
Abbreviations:
ds, double-stranded.
h, human.
HMG protein, high-mobility group protein.
m, mouse.
mtRNAP, mitochondrial RNA
polymerase.
mtTFA or mtTFB, mitochondrial transcription factor A or B.
rpHPLC, reversed-phase HPLC.
sc, Saccharomyces
cerevisiae.
xl, Xenopus
laevis.
| |
ACKNOWLEDGMENTS |
We are very grateful to Dr. Thomas Lisowsky for helpful hints
concerning the denaturation-renaturation chromatography and to Dr.
Richard Reinhard for generously providing excellent support and rpHPLC
facilities. We also thank Dr. Hans Peter Braun and Dr. Lutz Grohmann
for many suggestions about protein-purification techniques.
| |
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Abstract
Introduction