From the Graduate School of Agricultural Sciences, Nagoya University, Nagoya 464-8601, Japan
Received for publication, March 20, 2001
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The DE1 sequence is a
cis-regulatory element necessary and sufficient for light
down-regulated and dark-inducible expression of the pea GTPase
pra2 gene. This sequence does not show any sequence similarity to the previously reported ones involved in light-regulated gene expression. A one-hybrid screen isolated a cDNA encoding a
DNA-binding protein, named DF1, with specificity for the DE1 sequence
5'-TACAGT. DF1 has domains similar to the trihelix DNA-binding domain
found in the GT-1 and GT-2 proteins, which are plant transcription factors. The DE1-binding domain of DF1 is most similar to the carboxyl-terminal trihelix domain of the rice GT-2 protein with specificity for the GT2 sequence 5'-GGTAATT, which is also necessary for dark-inducible expression of the rice phyA gene. An
electrophoretic mobility shift assay showed that this DNA-binding
domain specifically binds to two types of DNA sequences, DE1 and GT2.
Additionally, using DF1/GT-1 chimeras, we show that the second and
third helices of the trihelix DNA-binding domain of DF1 are responsible
for this dual DNA binding specificity. Our results show that DF1
has specificity for the two distinct cis-regulatory
elements, both important for light down-regulated and dark-inducible
gene expression in higher plants.
Light is one of the most important signals affecting plant gene
expression (1). Light signals regulate the transcription of a number of
genes in positive and negative manners. The mechanisms of
light-enhanced transcription have been extensively studied (1). Various
cis-regulatory elements and trans-acting factors responsible
for light-enhanced gene expression have been characterized (1). In
contrast, the mechanism of light down-regulated transcription is less
clear. Only a few cis-regulatory elements and trans-acting factors responsible for down-regulation have been reported. For example, the GT2 (5'-GGTAATT) sequence in the light down-regulated rice
phyA gene is necessary for dark-activation of this gene (2). Affinity screening using GT2 sequences has been used to isolate the
DNA-binding protein, named GT-2 (2). The GT-2 protein is a member of
the trihelix DNA-binding proteins (3), which are considered to be
specific to plants and have domain(s) containing three Previously, we reported that a small GTPase gene in pea,
pra2, which belongs to the YPT/rab family, is one of the
genes whose expression is down-regulated by photoreceptor phytochrome
(4). The pra2 gene is mainly expressed in the growing zone
of etiolated epicotyls, and its expression is repressed when the plant
is illuminated (5). The DE1 sequence (5'-GGATTTTACAGT) in the
pra2 gene is another cis-regulatory element
necessary for light down-regulated and dark-enhanced expression in
plants (6). Gain-of-function analysis has shown that the DE1 sequence
is sufficient to confer dark-inducible and light down-regulated
expression to a minimal promoter (7). This sequence does not show any
sequence similarity to the previously reported ones involved in
light-regulated gene expression. In addition, to our knowledge, DE1 is
the first discovered cis-regulatory element that is both
necessary and sufficient for light regulation. Physiological and
genetic analyses have shown that the DE1 sequence receives the signals
from phytochrome A, phytochrome B, and blue-light photoreceptors (7).
Therefore, the DE1 sequence should be a useful tool for studying the
molecular mechanism involved in light down-regulated gene expression in plants. In this study, we identified a cDNA whose product binds to
the DE1 sequence. We found that this protein is a member of the
trihelix DNA-binding proteins and binds to two distinct
cis-regulatory elements, DE1 and GT2, both important for
light down-regulated and dark-inducible expression in plants.
Reporter Constructs for the Library Screen--
The methods for
the creation of reporter constructs were described previously (7). For
the first screening, the pHISi reporter plasmid
(CLONTECH, Palo Alto, CA) containing five tandem
copies of the 18-bp1
sequence, which contains the DE1 sequence with neighboring 3 bp at both
ends, was used. For the second screening, the pLacZi reporter plasmid
(CLONTECH) containing nine tandem copies of the 18-bp sequence, which contains the DE1 sequence with neighboring 3-bp
at both ends, was used. For the third screening, the pLacZi reporter
plasmid containing three tandem copies of the 20-bp sequence, which
contains the DE1 sequence with neighboring 4 bp at both ends, was used.
The reporter constructs were integrated into the yeast
Saccharomyces cerevisiae YM4271
(CLONTECH).
Preparation of the cDNA Library--
Total RNA was prepared
from the growing zones of pea (Pisum sativum cv. Alaska)
stems (between 0 and 1 cm from the top of the hook) that had been grown
in the dark for 7 days at 25 °C as described elsewhere (8).
Poly(A)+-RNA was extracted by the Poly(A)Tract mRNA
isolation system (Promega, Madison, WI). Complementary DNA was
synthesized with the HybriZAP-2.1 two-hybrid cDNA synthesis kit
(Stratagene, La Jolla, CA), cloned into the EcoRI and
XhoI sites of HybriZAP-2.1 or Screening of the cDNA Library--
The histidine yeast
reporter strain was transformed with a pea cDNA library by the
LiAc/polyethylene glycerol method. Approximately 1.5 × 107 cDNA plasmids were screened. Based on their large
colony size and rapid growth, about 500 histidine-positive clones were
selected. Plasmids were recovered and electroporated into the
Escherichia coli strain DH10B (Life Technologies, Inc.).
Plasmids were rescreened by transforming the second lacZ
reporter strain. The filter-replica method using X-gal was used to
confirm Production of Recombinant DF1 Protein--
The protein
expression vector used in this study was pGEX-4T-3 (Amersham Pharmacia
Biotech) or the pET-16b vector (Novagene). Procedures for the
production and purification of a fusion polypeptide were carried out as
suggested by the manufacturer. The polypeptide containing the trihelix
DNA-binding domain (from 519 to 651) of the pea DF1 protein was
expressed in E. coli as a decahistidine-tagged (His10 tag) protein or a glutathione
S-transferase (GST)-fused protein. The GST-fused GT-1
protein contains the Arabidopsis GT-1 sequence (from 65 to
195). DF1/GT-1 chimeras were created by the overlap extension method
(9). The GST-fused chimera protein, GT-1helix 1-DF1helices 2-3, contains the
Arabidopsis GT-1 sequence (from 65 to 108) and the pea DF1
sequence (from 563 to 651). Another GST-fused chimera protein,
DF1helices 1-2-GT-1helix 3, contains the pea
DF1 sequence (from 519 to 578) and the Arabidopsis GT-1
sequence (from 125 to 195).
Electrophoretic Mobility Shift Assay--
The electrophoretic
mobility shift assay (EMSA) was performed by the method previously
described (6). The probes used in this study, DE1 and GT2, are shown in
Figs. 3A and 4A, respectively. The recombinant
protein (5 pmol) was mixed in 20 µl of the binding buffer (6)
containing 2 µg of poly(dI-dC)-poly(dI-dC), bovine serum albumin (500 µg/µl) and competitor DNA. The competitor DNAs used in this study
are listed in Figs. 3A and 4A. The protein-DNA complex was formed by incubating at 25 °C for 20 min with 10,000 cpm
of 32P-labeled probe (4 fmol). Electrophoresis was
conducted at 4 °C in a 5% polyacrylamide Tris/borate/EDTA gel
containing 10% glycerol. The gel was dried and subjected to autoradiography.
Isolation and Sequence Analysis of cDNA Encoding a DNA-binding
Protein with Specificity for the DE1 Element--
The yeast one-hybrid
strategy (10) was used to screen for pea cDNA encoding for protein
that binds to the DE1 sequence. Tandem copies of the 18-bp sequence
containing the 12-bp DE1 sequence with a neighboring 3 bp at each end
were subcloned into the upstream region of HIS3 and
lacZ reporter genes. About 500 histidine-positive clones
were selected from 1.5 × 107 transformants (Fig.
1A). We rescreened plasmids
recovered from histidine-positive clones by transforming the
lacZ reporter strain and obtained three plasmids showing
lacZ-positive phenotypes (Fig. 1B). To exclude
the cDNAs whose products bind to the joint sequence between the 18 bp sequences, we rescreened the plasmids using the reporter constructs
having tandem copies of the 20-bp sequence containing the DE1 sequence
with a neighboring 4 bp at each end. Finally, one plasmid showed a
lacZ-positive phenotype (Fig. 1C). To isolate
longer cDNAs, this cDNA was used to screen a
The gene, designated as DF1, contains an open reading frame
of 682 amino acid residues. A sequence similarity search revealed that
DF1 has domains similar to the trihelix DNA-binding domain (3) found in
GT-1 (11, 12), GT-2 (2, 13), and GTL1 (14) proteins. GT-1 and GT-2 are
DNA-binding proteins that recognize light-responsive
cis-regulatory elements and are homologous within trihelix
DNA-binding domains, which contain three DF1 Is a DNA-binding Protein with Specificity for the DE1
Element--
To analyze the DNA-binding specificity of the DF1
protein, we conducted an EMSA using a 31-bp synthetic DNA probe
containing the single DE1 sequence (Fig.
3A, WT). We focused
only on the carboxyl-terminal trihelix domain of DF1 because a
one-hybrid screen first isolated this DNA-binding domain, and
studies of both domains would have been complicated. We expressed the
carboxyl-terminal trihelix DNA-binding domain of DF1 in E. coli as a His10 tag or a GST-fused protein. The
addition of the His10 tag DF1 protein to the binding
reaction mixture showed the retarded band(s) of the DNA-protein
complexes (Fig. 3B, lane 2). The addition of
the GST-fused DF1 protein to the binding reaction mixture also showed the retarded bands of DNA-protein complexes (Fig. 3B,
lane 3). Because it is easy to produce sufficient amounts of
recombinant GST-fused protein and it is difficult to dissolve the
His10 tag DF1 protein in solution, we used the GST-fused
DF1 protein in the following experiments. The results were essentially
the same as those of the experiments using the His10 tag
protein (data not shown).
To test whether the observed binding was specific to the DE1 sequence,
we carried out competition experiments. The competitor sequences are
shown in Fig. 3A. The addition of a 400-fold wild-type competitor diminished the retarded band (Fig. 3B, lane
4, WT). The MT competitor contained the three nucleotide changes,
which could not bind to the nuclear factors with specificity for the DE1 sequence (6). A gain-of-function analysis using the 3-bp mutated
constructs fused to the minimal promoter has shown that this construct
does not have the ability for light down-regulation (7). The EMSA
showed that the MT competitor did not diminish the retarded band (Fig.
3B, lane 5). These data indicate that the DNA
binding specificity is consistent with the results of the EMSA using
nuclear extracts (6) and the gain-of-function analysis (7).
To determine the core-binding site of the DF1 protein, we carried out
competition experiments using the 6-bp mutated competitors (Fig.
3A, LS2-LS4). In our previous work (6), we
identified the DE1 element using the 6-bp mutated constructs by
transient assay analysis. The LS3 construct (Fig. 3A) did
not show red-light down-regulation. The LS2 construct (Fig.
3A) affected red-light down-regulation. This analysis
indicates that the 12-bp sequence, designated DE1 (5'-GGATTTTACAGT),
mediates phytochrome down-regulation and that the 6-bp sequence
(5'-TACAGT) is the core region for down-regulation. The addition of a
400-fold LS3 competitor did not diminish the retarded band (Fig.
3B, lane 7). The addition of an LS2 competitor
diminished the band slightly (Fig. 3B, lane 6),
whereas the addition of an LS4 competitor diminished the band greatly
(Fig. 3B, lane 8). These results show that
recombinant DF1 binds the DE1 sequence and the core-binding site is the
sequence 5'-TACAGT. Thus, these results are very consistent with those of the transient assay analysis (6).
To determine the DF1-binding site more precisely, we carried out
competition experiments using the pairwise-mutated competitors (Fig.
3A, MT1-MT9). The addition of MT4, MT5, and MT6
competitors, which correspond to the core region of the DE1 element,
did not diminish the retarded band (Fig. 3B, lanes
12-14). The addition of the MT2 and MT3 competitors diminished
the band slightly (Fig. 3B, lanes 10 and
11), and the addition of other competitors diminished the
band greatly (Fig. 3B, lane 9 and lanes
15-17). These results show that the sequence 5'-TACAGT is the
core-binding site of the DF1 protein, and its surrounding sequence is
slightly involved in the binding of the DF1 protein.
DF1 Is a DNA-binding Protein with Specificity for the GT2
Element--
The DE1-binding domain shows sequence similarities to
many trihelix DNA-binding domains. Among these, the binding sequences of GT-1 (11, 12) and GT-2 (2, 13) proteins have been characterized. The
GT1 element (5'-GTGTGGTTAATATG) of pea rbcS-3A is the binding site of tobacco GT-1. (The core-binding sequence is
underlined.) The GT2 (5'-TGGCGGTAATTAAC) and the GT3
(5'-TCGAGGTAAATCCG) sequences of rice phyA are
the binding sites of carboxyl-terminal and amino-terminal trihelix
DNA-binding domains, respectively, of the rice GT-2 protein (13). Thus,
the DF1 binding sequence (5'-TACAGT) is clearly different from the
binding sequences of GT-1 and GT-2 proteins. The DE1-binding domain of
the DF1 protein is most similar to the GT2 element-binding domain of
the rice GT-2 protein. To determine whether DF1 has the ability to bind to the binding sites of these trihelix DNA-binding proteins, we carried
out competition experiments using competitors containing the above
binding sequences (Fig. 3A, GT1-GT3). The
addition of GT1 and GT3 competitors did not diminish the retarded band
(Fig. 3B, lanes 18 and 20). However,
the addition of a GT2 competitor diminished the band (Fig.
3B, lane 19). Thus, these results suggest that
DF1 has the ability to bind to the GT2 element.
The complementary sequence of the GT2 probe
(5'-TGGCGGTAATTAAC) is 5'-GTTAATTACCGCCA. In this
complementary sequence, the sequence 5'-TACCGC is partly similar to the
core DE1 sequence 5'-TACAGT. Therefore, the DF1 protein might bind to
this sequence and not to the GT2 core sequence. To determine the DNA
binding specificity to the GT2 sequence, we also conducted the EMSA
using a 31-bp synthetic DNA probe containing the GT2 sequence (Fig. 4A, GT2) and the
pairwise-mutated competitors (Fig. 4A). The addition of the
GST-fused DF1 protein to the binding reaction mixture showed the
retarded bands of the DNA-protein complex (Fig. 4B,
lane 2). The addition of MT12, MT13, MT 14, and MT15
competitors, which correspond to the core region of the GT2 element,
did not diminish the retarded bands (Fig. 4B, lanes
6-9). The addition of the MT11 competitors diminished the bands
slightly (Fig. 4B, lane 5), and the addition of
the GT2, MT10, MT16, and WT (containing the DE1 sequence) competitors
diminished the bands greatly (Fig. 4B, lanes 3-4
and lanes 10-11). This analysis showed that the
core-binding site of DF1 is 5'-GGTAATTA. This recognition specificity
of the DF1 protein is very similar to that of the GT-2 protein (2). Thus, these results again show that DF1 has the ability to bind specifically to the GT2 sequence, which is clearly different from the
core DE1 sequence.
Second and Third Helices of the DNA-binding Domain Are Responsible
for the Dual DNA Binding Specificity--
We are interested in knowing
which helices of the trihelix DNA-binding domain of DF1 are responsible
for the dual DNA binding specificity. In addition, it has not been
determined which helices of the trihelix DNA-binding domain of the
other related protein can recognize DNA sequences, although deletion
and mutational analyses have shown that the trihelix DNA-binding domain
of Arabidopsis GT-1 is essential for DNA binding (16). To
determine the mode of action for recognizing two distinct DNA
sequences, we conducted the EMSA using DF1/GT-1 chimeras (Fig.
5). Fig. 5A shows the
recombinant proteins used in this study. Both DF1 and
GT-1helix 1-DF1helices 2-3 proteins were
able to bind to two types of sequences, the GT2 and DE1 sequences (Fig.
5B, lanes 2, 3, 7, and 8), although
the DF1 protein bound to these sequences more strongly. Each of two types of proteins could have similar affinities to the GT2 and DE1
sequences. In contrast, the
DF1helices 1-2-GT-1helix 3 protein bound to
neither of two types of sequences (Fig. 5B, lanes
4 and 9). The GT-1 protein bound to both sequences only weakly (Fig. 5B, lanes 5 and 10).
Thus, the second and third helices of the trihelix domain are
responsible for the binding to the DE1 and GT2 sequences.
The creation of the chimera protein may affect the DNA binding
specificity. To determine the DNA binding specificity of the DF1helices 1-2-GT-1helix 3 protein to both
sequences, we conducted the EMSA using the pairwise-mutated competitors. The addition of MT12, MT13, MT14, and MT15 competitors to
the binding reaction mixture containing the labeled GT2 probe did not
diminish the retarded bands (Fig. 5C, lanes
5-8). On the other hand, the addition of GT2, MT11, and MT16
competitors diminished the bands slightly (Fig. 5C,
lanes 3, 4, and 9). Thus, although the
specificity to the sequence was reduced markedly, the core GT2
sequence, 5'-GGTAATTA, could be the core-binding site of the DF1helices 1-2-GT-1helix 3 protein.
Similarly, the addition of MT4, MT5, and MT6 competitors to the binding
reaction mixture containing the labeled DE1 probe did not diminish the
retarded bands (Fig. 5C, lanes 14-16). On the
other hand, the addition of WT, MT3, and MT7 competitors diminished the
bands slightly (Fig. 5C, lanes 12, 13,
and 17). These results show that the DE1 sequence 5'-TACAGT
is the core-binding site of the
DF1helices 1-2-GT-1helix 3 protein. Thus,
this chimera protein can recognize two distinct DNA sequences, DE1 and
GT2, indicating that second and third helices of the DNA-binding domain
are responsible for the dual DNA binding specificity.
The carboxyl-terminal DNA-binding domain of DF1 can recognize two
distinct cis-regulatory elements, DE1 and GT2, both
important for light down-regulated and dark-inducible gene expression
in higher plants. This is an especially exquisite mechanism as DF1 protein transduces information from the light signal to the two effectors, DE1 and GT2. Plants must have acquired this mechanism during
the course of evolution. One possible explanation for this evolved
mechanism is that ancestral DF1 protein could bind to only one
recognition sequence and the subsequent discontinuous change(s) in the
amino acid sequence of the DNA-binding domain resulted in the dual DNA
binding specificity. Another possibility is that continual changes in
both the DNA-binding domain and its recognition sequences eventually
resulted in the dual DNA binding specificity.
In the current study, we have described a DNA-binding protein having a
domain that recognizes two distinct DNA sequences, although both are
cis-regulatory elements having similar functions. Interestingly, two recognition sequences show no or little sequence similarities. To date, cases in which one DNA-binding protein has the
specificity for distinct, although related, sequences have been
reported. For example, the DNA-binding protein MYB.Ph3, a member of the
MYB proteins in Petunia, binds to two types of sequences:
5'-A(a/D)(a/D)C(G/C)GTTA (where a/D is A, G, or T, A as the preferred
base) and 5'-AGTTAGTTA (17). In contrast, murine c-MYB only binds to
the former sequence. A single residue substitution in MYB.Ph3 caused a
switch from the dual DNA binding specificities to the c-MYB specificity
(17). In addition, c-MYB with the reciprocal substitution gained
MYB.Ph3 specificity (17). Interestingly, the trihelix DNA-binding
domain may be distantly related to the Myb DNA-binding domain (15).
Single or multiple residue substitutions in the trihelix DNA-binding
domain from the ancestral protein might cause dual DNA binding
specificity for two distinct and unrelated cis-regulatory elements.
The dual DNA binding specificity might be the common feature of the
trihelix DNA-binding protein. Arabidopsis GT-1 binds not only to the GT1 sequence 5'-GGTTAA but also to the repeated GATA sequences (16). However, the precise characteristics of the protein
have not been determined because the precise specificity to the GATA
sequence has not been determined and these two recognition sequences
may be related.
Affinity screening using GT-1 binding sites has isolated the GT1
protein (11, 12). These studies have postulated that three putative In the current study, we added a characterized member of trihelix
DNA-binding proteins. The carboxyl-terminal DNA-binding domain of DF1
can bind to two distinct cis-regulatory elements, DE1 and
GT2, both responsible for light down-regulated and dark-inducible gene
expression in higher plants. The rice GT-2 protein binds to the GT2
sequence, which is necessary for dark-inducible expression of the rice
phyA gene. As the GT-2 protein shares a high degree of
similarity to DF1, the GT-2 protein might also bind to the DE1
sequence. Another trihelix DNA-binding protein, GT-1, can bind to the
GT1 sequence, which is necessary but not sufficient for light-induced
expression of the pea rbcS-3A gene (11, 12). Thus, the
trihelix DNA-binding proteins could be generally important for
light-regulated gene expression. In addition, the current study
emphasizes the importance of twin trihelix DNA-binding proteins for
light down-regulated and dark-inducible gene expression in plants.
Arabidopsis, the genome of which was almost completely sequenced (18), contains more than 10 trihelix DNA-binding proteins (several of which are listed in Refs. 4 and 16). Further characterization of the trihelix DNA-binding proteins, especially of
twin trihelix DNA-binding proteins including DF1 protein, should be an
important task for the research of light-regulated gene expression in plants.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
helices
involved in DNA binding. Although the GT-2 protein is one of the well
characterized plant DNA-binding proteins, the function of its binding
site, the GT2 sequence, is less clear because there has been no report
concerning a gain-of-function analysis using the GT2 sequence fused to
the minimal promoter. Therefore, combinatorial interaction between the
GT2 element and the other element may be required for dark activation.
Except for the GT2 protein, we do not know of a DNA-binding protein
with specificity for the cis-regulatory element involved in
light down-regulated gene expression.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
ZapII, and packaged using
the MaxPlax packaging extract (Epicentre Technologies, Madison, WI).
The plasmid library was constructed by excising from the HybriZAP
library by infection with the helper phage ExAssist (Stratagene). The
resulting HybriZAP cDNA library contained ~8 × 106 independent cDNAs.
-galactosidase activities. Three plasmids showed the blue
color. These plasmids were rescreened by transforming the third
lacZ reporter strain. One plasmid showed the blue color. To
isolate the full-length cDNA, a
ZapII cDNA library was
screened using a part of this cDNA insert as a probe. The gene was
named DF1. The accession number for the sequence reported in this
article is AB052729.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
ZAPII cDNA
library. We further characterized the longest cDNA of the isolated
clones.
View larger version (21K):
[in a new window]
Fig. 1.
Schematic diagram of one-hybrid
strategy.
helices involved in DNA
binding. Fig. 2A shows the
schematic representation of DF1 and other related proteins. Among
trihelix DNA-binding proteins, DF1 is the most similar to proteins
having twin trihelix DNA-binding domains, such as GT-2 and GTL-1
proteins. These twin trihelix DNA-binding proteins differ structurally
from GT-1 because GT-1 has only one trihelix DNA-binding domain. The
DF1 protein also has twin trihelix domains. In addition to having two
conserved DNA-binding domains, GT-2, GTL-1, and DF1 proteins each have
an additional conserved domain, a central domain, which is located between two DNA-binding domains. However, except for the three conserved domains, the amino acid sequences of these twin trihelix DNA-binding proteins are not conserved. Fig. 2B shows the
multiple alignments among several trihelix DNA-binding proteins. As
recent in silico analysis has postulated that the trihelix
domain is distantly related to the Myb DNA-binding domain (15), the
alignment includes the c-Myb DNA-binding domains. All these proteins
have well conserved amino acid residues. Among Arabidopsis
proteins, the amino acid sequence of DF1 is most similar to that of the protein predicted from the BAC clone F7O12 (Fig. 2C). Thus,
DF1 is not orthologous to GT-2 and GTL-1 proteins. However, proteins having twin trihelix DNA-binding domains, such as DF1, GT-2, and GTL-1,
may have overlapping functions because they share a high degree of
similarity. The orthologous Arabidopsis gene is located adjacent to the GT-2 gene, in the opposite orientation, on
chromosome 1.
View larger version (41K):
[in a new window]
Fig. 2.
A, schematic representation of
the sequence comparison of pea DF1, tobacco GT-1, and rice GT-2
proteins. Black boxes represent the trihelix DNA-binding
domain. Gray boxes represent the central domain conserved
among twin trihelix DNA-binding proteins. The amino acid identities
among the conserved domains are shown. The arrow indicates
the first isolated cDNA. B, amino acid sequence
comparisons of several trihelix DNA-binding proteins and c-Myb
DNA-binding domains. Gray boxes represent the well conserved
amino acids of both classes of proteins. helix regions of c-Myb
DNA-binding domains are underlined based upon the solution structure
(19). Species abbreviations are as follows: Ps, Pisum
sativum; At, Arabidopsis thaliana; Os, Oryza
sativa; Nt, Nicotiana tabacum; Mm, Mus
musculus. N, amino-terminal trihelix domain;
C, carboxyl-terminal trihelix domain. C, location
of DF1 and GT-2 genes on Arabidopsis
chromosome 1. Black and white boxes designate
exon and intron sequences, respectively; the boxes above the
line are translated to the right, and those below
the line are translated to the left.
View larger version (63K):
[in a new window]
Fig. 3.
Determination of the DNA sequence bound to
the DF1 protein using the DE1 probe. A, nucleotide
sequences of various competitors used to define the DF1-binding site.
The positions of the mutated nucleotide are shaded. The
core-binding site of the DF1 protein is underlined.
B, EMSA using various competitors.
View larger version (33K):
[in a new window]
Fig. 4.
Determination of the DNA sequence bound to
the DF1 protein using the GT2 probe. A, nucleotide
sequences of various competitors used to define the DF1-binding site.
The positions of the mutated nucleotide are shaded. The
core-binding site of the DF1 protein is underlined.
B, EMSA using various competitors. The competitors
WT and MT are shown in Fig. 3.
View larger version (32K):
[in a new window]
Fig. 5.
Determination of the DNA sequences bound to
the DF1/GT-1 chimera protein. A, schematic
representation of the recombinant proteins used in this study. These
proteins were fused to the C terminus of GST. B, EMSA using
various recombinant proteins and the GT2 or DE1 probe. The proteins
used in this study are shown in A. Lanes 1 and
6 do not contain recombinant protein. C, EMSA of
the DF1helices 1-2-GT-1helix 3 protein
using various competitors and the GT2 or DE1 probe. The competitors
used in this study are shown in Figs. 3 and 4.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
helices (trihelix domain) in GT-1 might be involved in DNA binding.
Subsequent deletion analyses of GT-1 have shown that the trihelix
DNA-binding domain of GT-1 is certainly involved in DNA binding (16).
However, it has not been determined which helices are responsible for
DNA binding. Using DF1/GT-1 chimeras, the current study showed that
second and third helices of the trihelix DNA-binding domain of DF1 are
responsible for specific DNA binding. This mode of action is similar to
those of the other DNA-binding proteins containing three helices, such
as the Myb DNA-binding domain and the homeodomain. A recent in
silico study has postulated that the trihelix DNA-binding domain
is distantly related to the Myb DNA-binding domain (15). Thus, this
postulation is consistent with our results using DF1/GT-1 chimeras.
![]() |
ACKNOWLEDGEMENT |
---|
We thank Dr. K. Hiratsuka for useful suggestions.
![]() |
FOOTNOTES |
---|
* This work was supported by Grants-in-aid for Encouragement of Young Scientists (no. 12760225) and for Priority (no. 13017210) (to Y. N.) from the Japanese Ministry of Education, Culture, Science, Sports, and Technology. This work was also supported by the Japan Society for the Promotion of Science, Research for the Future Program Grants No. JSPS-RTFT.96L006012 (to Y. S.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AB052729.
To whom correspondence should be addressed: Graduate School
of Agricultural Sciences, Nagoya University, Nagoya 464-8601, Japan.
Tel.: 81 52 789 4168; Fax: 81 52 789 4296; E-mail:
nagano@agr.nagoya-u.ac.jp.
§ Recipient of research fellowships from the Japan Society for the Promotion of Science for Young Scientists.
Published, JBC Papers in Press, April 4, 2001, DOI 10.1074/jbc.M102474200
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
bp, base pair;
X-gal, 5-bromo-4-chloro-3-indolyl--D-galactopyranoside;
EMSA, electrophoretic mobility shift assay;
WT, wild type;
GST, glutathione S-transferase;
His10 tag, decahistidine-tagged.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Terzaghi, W. B., and Cashmore, A. R. (1995) Annu. Rev. Plant Physiol. Plant Mol. Biol. 46, 445-474[CrossRef] |
2. | Dehesh, K., Bruce, W. B., and Quail, P. H. (1990) Science 250, 1397-1399[Medline] [Order article via Infotrieve] |
3. | Zhou, D.-X. (1999) Trends Plant Sci. 4, 210-214[CrossRef][Medline] [Order article via Infotrieve] |
4. | Yoshida, K., Nagano, Y., Murai, N., and Sasaki, Y. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 6636-6640[Abstract] |
5. | Nagano, Y., Okada, Y., Narita, H., Asaka, Y., and Sasaki, Y. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 6314-6318[Abstract] |
6. |
Inaba, T.,
Nagano, Y.,
Sakakibara, T.,
and Sasaki, Y.
(1999)
Plant Physiol.
120,
491-500 |
7. |
Inaba, T.,
Nagano, Y.,
Reid, J. B.,
and Sasaki, Y.
(2000)
J. Biol. Chem.
275,
10723-19727 |
8. | Nagano, Y., Murai, N., Matsuno, R., and Sasaki, Y. (1993) Plant Cell Physiol. 34, 447-455[Medline] [Order article via Infotrieve] |
9. | Ho, S. N., Hunt, H. D., Horton, R. M., Pullen, J. K., and Pease, L. R. (1989) Gene (Amst.) 77, 51-59[CrossRef][Medline] [Order article via Infotrieve] |
10. | Wang, M.-M., and Reed, R. R. (1993) Nature 364, 121-126[CrossRef][Medline] [Order article via Infotrieve] |
11. |
Perisic, O.,
and Lam, E.
(1992)
Plant Cell
4,
831-838 |
12. |
Gilmartin, P. M.,
Memelink, J.,
Hiratsuka, K.,
Kay, S. A.,
and Chua, N.-H.
(1992)
Plant Cell
4,
839-849 |
13. | Dehesh, K., Hung, H., Tepperman, J. M., and Quail, P. H. (1992) EMBO J. 11, 4131-4144[Abstract] |
14. |
Smalle, J.,
Kurepa, J.,
Haegman, M.,
Gielen, J.,
Van Montagu, M.,
and Straeten, D. V.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
3318-3322 |
15. |
Nagano, Y.
(2000)
Plant Physiol.
124,
491-493 |
16. |
Hiratsuka, K.,
Wu, X.,
Fukuzawa, H.,
and Chua, N.-H.
(1994)
Plant Cell
6,
1805-1813 |
17. |
Solano, R.,
Fuertes, A.,
Sanchez-Pulido, L.,
Valencia, A.,
and Paz-Ares, J.
(1997)
J. Biol. Chem.
272,
2889-2895 |
18. | The Arabidopsis Genome Initiative. (2000) Nature 408, 796-815[CrossRef][Medline] [Order article via Infotrieve] |
19. | Ogata, K., Morikawa, S., Nakamura, H., Sekikawa, A., Inoue, T., Kanai, H., Sarai, A., Ishii, S., and Nishimura, Y. (1994) Cell 79, 639-648[Medline] [Order article via Infotrieve] |