From the Institute of Botany, Academia Sinica,
Taipei 115 and the ¶ Institute of Biochemical Sciences, National
Taiwan University, Taipei 106, Taiwan, Republic of China
Received for publication, October 23, 2000, and in revised form, January 10, 2001
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ABSTRACT |
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A gene (AtTRP1) encoding a telomeric
repeat-binding protein has been isolated from Arabidopsis
thaliana. AtTRP1 is a single copy gene located on chromosome 5 of
A. thaliana. The protein AtTRP1 encoded by this gene is not
only homologous to the Myb DNA-binding motifs of other telomere-binding
proteins but also is similar to several initiator-binding proteins in
plants. Gel retardation assay revealed that the 115 residues on the C
terminus of this protein, including the Myb motif, are sufficient for
binding to the double-stranded plant telomeric sequence. The isolated DNA-binding domain of AtTRP1 recognizes each telomeric repeat centered
on the sequence GGTTTAG. The almost full-length protein of AtTRP1 does
not form any complex at all with the DNA fragments carrying four or
fewer GGTTTAG repeats. However, it forms a complex with the sequence
(GGTTTAG)8 more efficiently than with the sequence (GGTTTAG)5. These data suggest that the minimum length of a
telomeric DNA for AtTRP1 binding consists of five GGTTTAG repeats and
that the optimal AtTRP1 binding may require eight or more GGTTTAG
repeats. It also implies that this protein AtTRP1 may bind in
vivo primarily to the ends of plant chromosomes, which consist of
long stretches of telomeric repeats.
Telomeres, the specialized nucleoprotein structures at the ends of
eukaryotic chromosomes, are essential for the maintenance of chromosome
integrity (1, 2). The telomeric DNA in most eukaryotic chromosomes
consists of tandemly repeated sequences (3); the sequence TTTAGGG has
been identified in the telomeres of most plant species (4, 5). The
length of plant telomeres varies from a few kilobase pairs
(kbp)1 in
Arabidopsis (6) to a few hundred kbp in Nicotiana
(7). A study in maize suggested that variation of telomere length among strains is controlled by multiple genes (8). Studies in yeast and
mammalian cells revealed that the telomere length is regulated by
multiple factors including telomerase and telomeric repeat-binding proteins (TRPs) (9). Telomerase is a reverse transcriptase that uses an
internal RNA moiety as a template for the extension of G-rich strand at
the ends of DNA molecules (10) under the control of
telomerase-associated proteins (11-14). In human cells, telomerase
activity and telomere length are tissue-specific and under
developmental control (15). In plants, the telomerase activity is also
correlated with the cellular proliferation capacity (16-18). However,
telomere length remains unchanged during development in some species
but shortens dramatically in others (19-21). This implies that
telomerase may not be the sole factor in controlling telomere length in
plants. Disruption of the gene for the telomerase catalytic subunit in
Arabidopsis only results in a slow loss of telomeric DNA
(22), indicating that the telomerase-deficient plant cells may have
other, unknown, mechanism(s) to prevent telomeres from shortening rapidly.
TRPs bind directly to either single-stranded or double-stranded
telomeric DNA. The former interact with single-stranded 3' extension of
the extreme termini and are important for the maintenance of telomere
length by restricting access of telomerase to chromosome termini (23,
24). Double-stranded telomeric repeat-binding proteins, such as Rap1p
in budding yeast (25), Taz1p in fission yeast (26), and hTRF1 (27) and
hTRF2 (28, 29) in human cells, have various functions. The Rap1p, an
abundant telomeric protein in budding yeast, inhibits telomere
elongation (30) and controls the expression of numerous genes involved
in cell growth (31). The Taz1p is not only a factor in the negative regulation of telomere length (26) but is also required for meiotic
telomere clustering and genetic recombination in fission yeast
(32-34). In human cells, telomere length is negatively regulated by
hTRF1 (35) and also by hTRF2 (36) which is also required for
maintaining telomere integrity (37). Lack of hTRF2 induces apoptosis in
some cell lines (38). These results suggest that TRPs play a crucial
role in telomere and cellular metabolism.
To explore the structure and function of TRPs in plants, efforts have
been made to characterize the factors that can bind to plant telomeric
sequence. These studies included the detection of double-stranded
telomeric DNA-binding protein in maize and Arabidopsis crude
extracts (39), the identification of factors that bind to
single-stranded G-rich telomeric repeats in rice and mung bean nuclear
extracts (40, 41), and the characterization of an
Arabidopsis protein (ATBP1) that binds to the G-rich as well
as to the double-stranded telomeric sequence (20, 42). A recent paper
described the cloning of a rice cDNA that encodes a protein (RTBP1)
that contains a C-terminal telomeric DNA-binding domain (43). The
isolated DNA-binding domain of RTBP1 bound specifically to the duplex
oligonucleotide sequence (TTTAGGG)4; however, no evidence
was shown that the full-length protein of RTBP1 can distinguish DNA
fragments carrying long arrays of telomeric repeats from those with
short ones. Therefore, it remains unclear whether the RTBP1 is bound to
the long stretches of telomeric repeats at chromosome ends or to the
short telomeric repeats in interstitial regions of plant chromosomes.
Moreover, the exact core sequence in the plant telomeric repeat
recognized by the isolated DNA-binding domain of RTBP1 has not yet been
defined. The mode of interaction between TRP and telomeric DNA in
plants therefore remains unclear.
We report here the cloning of an Arabidopsis gene
(AtTRP1) and the corresponding cDNA, which was expressed
in bacteria to produce a protein that specifically binds to the
double-stranded plant telomeric sequence. The DNA-binding domain of
AtTRP1 was defined, and the core sequence of each telomeric repeat
recognized by the isolated DNA-binding domain of AtTRP1 has been
determined. The minimum length of a telomeric DNA fragment bound by an
AtTRP1 protein has been defined. Our in vitro evidence
indicates that the AtTRP1 protein may be located primarily at the ends
of plant chromosomes.
Cloning of AtTRP1--
The oligonucleotide
(TTTAGGG)4 was placed upstream of the minimal
promoter in pHISi and pLacZi vectors (CLONTECH,
CA). These vectors were linearized and sequentially integrated into the
chromosomes of yeast strain YM4271 (MATa, ura3-52, his3-200,
ade2-101, lys2-801, leu2-3, 112, trp1-903, tyr1-501,
gal4- Production of AtTRP1 Protein in Bacteria--
A full-length
cDNA was obtained by digestion of partial cDNA clones (Fig.
1A) with appropriate enzymes, followed by sequential ligation. To generate cDNA subfragments oligonucleotides containing modified sequences of AtTRP1 were used as primers for PCR to
amplify the full-length cDNA: Bamp12 (GAATTTGGATCCGCAAGCTACC, derived from 1068-1089) and Bamp16 (GTACAACGGATCCTCTAAAAGCC, derived from 3685-3663) for Purification of AtTRP1 Derivatives--
The crude extract
containing the protein Gel Retardation Assay--
The single-stranded or duplex
oligonucleotides were end-labeled with digoxigenin-11-ddUTP (Roche
Molecular Biochemicals) and used as the probe for gel retardation
assay. Gel retardation assays were done in a volume of 20 µl
containing 1× DNA-protein binding buffer, 1 µg of poly[d(I-C)],
0.1 µg of poly-L-lysine, 1 pmol of digoxigenin-labeled
probe, and 5-10 µg of crude extracts or various amounts of the
purified proteins. For competition experiments, the binding reaction
was augmented with various amounts of competitor DNA. After 30 min at
25 °C, the free and bound DNA were separated by electrophoresis on 4 or 6% polyacrylamide gels in 0.5× TBE (44.5 mM Tris
borate, 1 mM EDTA, pH 8.0) at 4 °C, transferred to nylon
membranes, and detected by chemiluminescent reaction using CSPD
(C18H20CIO7PNa2) as
substrate (Roche Molecular Biochemicals).
Western Hybridization--
The purified protein Other Techniques--
Southern hybridization, amplification, and
screening of DNA libraries, bacterial transformation, plasmid
isolation, gel electrophoresis, and PCR were performed as described
(45). Yeast transformation and recovery of plasmids from yeast were
done as described by the manufacturer (CLONTECH, CA).
Cloning of AtTRP1--
The cDNA clones encoding TRPs in
A. thaliana were isolated by a yeast one-hybrid system.
Plasmids from all three positive transformants of yeast contained an
identical cDNA insert, 1-1, of 1.1 kbp in size (Fig.
1A). Overlapping cDNAs
1-9 and 1-20 were subsequently obtained using 1-1 as a probe. The
resulting sequence was extended further in the 5' direction by RACE on
the cDNA library from which the clone 1-1 was isolated. Alignment
of the cDNA clones and the RACE product, 1-21, yielded a
contiguous sequence spanning 2391 base pairs (bp). A genomic clone, G1,
containing AtTRP1 (Fig. 1A) was obtained from a
phage library using cDNA 1-1 as a probe. Southern hybridization
revealed that AtTRP1 is a single copy gene (data not shown).
Comparison of the sequences of the genomic and cDNA clones of
AtTRP1 indicated that this gene contains nine introns and
the first exon is untranslated (Fig. 1A). Each intron
contains the conserved dinucleotides GT and AG at 5' and 3' boundaries, respectively, suggesting that all the introns are of the U2-type (46).
A TATAAA sequence was located 63 bp upstream from the first exon and is
assumed to be the TATA box. The entire sequence of AtTRP1
matches exactly that of a genomic DNA fragment cloned from the
chromosome 5 of Arabidopsis thaliana
(GenBankTM AB025604).
Conceptual translation of the 2.4 kbp cDNA sequence reveals an open
reading frame of 578 amino acids that is predicted to encode a 65-kDa
protein with a pI of 8.4 (Fig. 1B). This protein, AtTRP1,
contains four clusters of basic residues (Fig. 1, B and C). The amino acid sequences in the first two clusters
(residues 22-36 and 213-219) are similar to those in the nuclear
localization signals (NLS) of the maize R gene product (47).
The sequence in the third cluster (residues 250-256) is homologous to
that in the NLS of SV40 T antigen, which facilitates the targeting of
proteins into nuclei of plants (48) and yeast (49). The presence of
multiple NLS suggests that AtTRP1 may be a nuclear protein. The last
cluster (residues 450-454), located next to the Myb motif, may help
the DNA-protein interaction since it is rich in positive charges and
close to the DNA-binding domain of AtTRP1 (see below). The sequence of
amino acids 466-520 is not only homologous to the Myb-related
DNA-binding motifs present in yeast and mammalian TRPs (Fig.
1D) but is also highly similar to the DNA-binding domain of
the rice protein RTBP1 (Fig. 2), suggesting that this region may be important for binding plant telomeric sequence. In addition to the Myb motif and NLS, AtTRP1 contains short clusters of acidic residues at its N-terminal end and
glutamine-rich regions on both sides of the Myb motif (Fig. 1B). Most interesting, AtTRP1 also contains contiguous
residues at several regions identical or similar to those of RTBP1 (43) and of three initiator-binding proteins in plants, including HPPBF1 (GenBankTM AF072536) from A. thaliana, BPF1 from
parsley (50), and IBP1 from maize (51) (Fig. 2). HPPBF1 and BPF1 bind,
respectively, to the promoters of the genes encoding the H protein of
glycine decarboxylase and phenylalanine ammonia lyase. IBP1 binds to
the promoter of the shrunken (Sh) gene involved
in carbohydrate metabolism.
The DNA-binding Domain of AtTRP1 Is Located at Its C
Terminus--
To map the DNA-binding domain of AtTRP1, cDNAs
encoding various truncated proteins (Fig.
3A) were cloned into the
vector pET3a and expressed in bacteria. Treatment of these cultures
with IPTG resulted in the accumulation of a unique protein in each
bacterial lysate (Fig. 3B). Since the DNA sequence analysis
has revealed that each cDNA construction only encodes a single
polypeptide, the unique protein in each lysate probably represents the
intact form of the corresponding truncated protein. The gel retardation assay of truncated proteins showed that the protein
Since the molecular weight of the protein The DNA-binding Domain of AtTRP1 Recognizes the Sequence
GGTTTAG--
To identify the core sequence in the telomeric DNA
recognized by the DNA-binding domain of AtTRP1, the protein The Minimum Length of a Telomeric DNA Bound by AtTRP1 Contains Five
GGTTTAG Repeats--
To explore the minimum length of the telomeric
DNA bound by AtTRP1, the protein
One or two complexes formed with the probes carrying five, six,
and eight GGTTTAG repeats. The weak complex I appeared occasionally in
the reactions containing the probes (GGTTTAG)5 and
(GGTTTAG)6 (Fig. 5B, lanes 7, 14, and
15), and always migrated the same distance. Complex II was
always the major one in all of the reactions and also always migrated
the same distance. Since the migration rate of DNA-protein complexes is
strongly influenced by the protein moiety in the complex (52) and since
these probes do not differ significantly from each other in size, it is
likely that these complexes with identical migration rates contain an
identical number of protein molecules associated with each probe. To
make sure that the protein moiety in both complexes is AtTRP1 Binds Specifically to DNA Fragments Carrying Long Arrays of
Plant Telomeric Repeats--
To determine the sequence specificity of
AtTRP1, the competition for the protein
The sequence in the DNA-binding domain of AtTRP1 is highly homologous
to those in the proteins HPPBF1, BPF1, and IBP1 (Fig. 2). Since the
sequence of the binding sites of IBP1 and BPF1is available (50, 51), we
examined whether AtTRP1 would recognize the binding sites of both
initiator-binding proteins. To address this question, we used the
oligonucleotides containing the binding sites of IBP1 (Fig. 6,
lanes 16-18) and BPF1 (Fig. 6, lanes 19-21) as
the competitors of (GGTTTAG)8 in the assay of complex
formation. Although both binding sites seemed to compete weakly with
the probe (GGTTTAG)8 in the complex formation, none of
these binding sites can form complex with the protein
Inspection of the nucleotide sequence of AtTRP1 has revealed
that a copy of plant telomeric repeat, GGGTTTA, is located next to the
putative TATA box of this gene. We have found that the isolated
DNA-binding domain of AtTRP1 could bind the duplex oligonucleotide AtTRPro which carries the sequence ATAGGCTTATAAAGGGTTTAAGCA (bases 67-90 of AtTRP1) around the putative TATA box of
AtTRP1 (data not shown). However, the competition experiment
shown in Fig. 6 (lanes 22-24) indicated that the protein
Gel retardation assay of cellular extracts followed by SDS-PAGE
analysis of the DNA-protein complex has identified a 67-kDa Arabidopsis protein (ATBP1) that forms a complex with the
duplex probe (TTTAGGG)4 (20, 42). Here we have shown that
the molecular mass of the almost full-length AtTRP1 protein is ~83
kDa, based on SDS-PAGE analysis (Fig. 5A), and this protein
does not form any complex with the probe (TTTAGGG)4 (data
not shown). The differences in both molecular mass and in the DNA
binding behavior of these two proteins suggest that there may be two
different telomere-binding proteins in A. thaliana.
Alternatively, ATBP1 could be one of the proteolytic products of AtTRP1
that have been found to form specific complexes with the probe
(TTTAGGG)4 during the purification of the protein The isolated DNA-binding domain of the rice protein RTBP1 formed only
three complexes with the probe (TTTAGGG)4 (43), whereas our
data showed that the corresponding polypeptide in AtTRP1 formed a
fourth complex with the identical probe at an extremely high molar
ratio of protein to DNA probe (Fig. 4B). Since both
polypeptides have highly similar sequences (Fig. 2), it is very likely
that the isolated DNA-binding domain of RTBP1 will form four complexes with the probe (TTTAGGG)4 in our experimental conditions.
In other words, the isolated DNA-binding domain of RTBP1 will behave
like that of AtTRP1 in the reaction with plant telomeric sequences. On the other hand, the duplex oligonucleotide (TTTAGGG)4
has been used by some as a probe for the isolation of plant TRPs from
nuclear extracts (20, 42). Our results have indicated that this probe is bound efficiently by some of the truncated proteins of AtTRP1 (Fig.
3 and 4) but not bound by the almost full-length molecule of the same
protein (data not shown), suggesting that the duplex oligonucleotide
(TTTAGGG)4 may not be the appropriate probe for the assay
of intact TRPs in plant cells.
The isolated DNA-binding domain of AtTRP1 can form complexes with the
probes carrying four or fewer telomeric repeats (Fig. 4),2 whereas the almost
full-length molecule of the same protein binds probes with at least
five GGTTTAG repeats (Fig. 5). Comparison of the DNA-binding properties
of the proteins Among the complexes formed between the protein The competition assay has shown that the protein Both AtTRP1 and hTRF1 proteins belong to the class of Myb proteins that
harbor only a single Myb motif (Fig. 1D). Protein hTRF1 has been shown to bind predominantly as a homodimer
to human telomeric DNA (56). It would be of interest to see whether
protein AtTRP1 would function as a dimeric protein and use a pair of
Myb-like DNA-binding domain to recognize DNA.
Our data has revealed a possible mode by which a telomeric
repeat-binding protein recognizes telomeric DNA in plants. The information about the features of the AtTRP1 protein allows us to
hypothesize a structure for protein-DNA complexes at plant telomeres
and propose possible roles for this protein in plant cells.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
512, gal80-
538,
ade5::hisG) to generate a reporter strain YR14.
Background LacZ activity in late logarithmic phase cells of YR14 was
low enough to be distinguished from a positive interaction. Low plating
density (108 cells per 150-mm plate) and 3-aminotriazole
(45 mM) were used to suppress growth on histidine-deficient
(
His) plates resulting from the low level of
(TTTAGGG)4-HIS3 reporter gene expression. An
Arabidopsis cDNA expression library cloned into the GAL4
activation domain (GAD) vector pGAD10 (CLONTECH,
CA) was amplified in Escherichia coli. Purified library DNA
(30 µg) was used to transform the YR14 strain. Positive clones were
selected on plates containing SD/
Leu/
His medium plus 45 mM 3-aminotriazole followed by assay for
-galactosidase activity (CLONTECH, CA). Plasmids in positive
clones were rescued by transformation into E. coli. The
insert in cDNA clone 1-1 was used as a probe to isolate overlapping
cDNA clones from an Arabidopsis cDNA library in
gt11 and genomic clones from an Arabidopsis genomic library in
EMBL3SP6/T7 (CLONTECH, CA). To obtain
the 5' end of full-length cDNA, rapid amplification of cDNA
ends (RACE) was performed by polymerase chain reaction (PCR) to amplify
the Arabidopsis cDNA library in pGAD10 with flanking
primer GADP1 (5'CTATTCGATGATGAAGATACCCCACCAAACCC3') and AtTRP1 primer
TBP12 (5'GGGACTCAAAGATCCCCG3'). The 5'-RACE PCR product was digested
with EcoRI and cloned into pUC18. All the cDNA and
genomic clones were sequenced in both strands using an ABI377 automatic
sequencer. Homology searches were made against sequences in the data
base of both GenBankTM and that generated by the
Arabidopsis Genome Initiative using BLAST.
12, Bamp3 (TATCTGGATCCGTTACTGATGA, derived from 2007-2028) and Bamp16 for
1, Bamp4
(GGTGATGGATCCGTGAGCACTTTAC, derived from 2441-2466) and Bamp16
for
2, Bamp5 (CTTGTGAGGATCCCTACCTGTG, derived from
2731-2753) and Bamp16 for
3, Bamp6 (GCAGCAGGATCCCAACGCAGAA, derived from 2942-2964) and Bamp16 for
4. These PCR products were
digested with BamHI and cloned into pET3a (44). To obtain DNA fragment
11, the 640-bp XbaI fragment was excised
form the DNA fragment
12, and the flanking fragments were ligated
and cloned into pET3a. To generate DNA fragment
31, Bamp3 was
coupled with Sacp3 (GTTGTGGAGCTCCTTAGCCGTG, derived from
3328-3306) and Bamp16 was coupled with Sacp1
(GGTCTGTTAGAGCTCTAAATGAATC, derived from 3469-3493) to generate,
respectively, DNA fragments
16 and
18 from the full-length
cDNA by PCR amplification. Fragments
16 and
18 were digested
with SacI and BamHI and then ligated with
BamHI-cut pET3a. All the truncated cDNAs were sequenced
to confirm that the sequences are correct. These truncated cDNAs in
pET3a were placed under the control of a bacteriophage T7 RNA polymerase promoter. Each construct was transformed in E. coli BL21(DE3) cells that were then induced by 0.4 mM
isopropyl-1-thio-
-D-galactopyranoside (IPTG) to
synthesize the truncated AtTRP1 (44). All the AtTRP1 derivatives
produced in bacteria contain an N-terminal addition of 14 amino acids
encoded by the DNA sequence of gene 10 of T7 phage fused to the
BamHI site in pET3a. After induction of the cultures with
IPTG for 2 h at 37 °C, the cells were spun down, washed twice
with buffer containing 50 mM Tris, pH 7.5, 0.2 M NaCl, and 5% glycerol, resuspended in 1× DNA-protein
binding buffer (20 mM Hepes, pH 7.6, 1 mM EDTA,
5% glycerol, 10 mM
(NH4)2SO4, 1 mM
dithiothreitol, 0.2% Tween 20, and 30 mM KCl) plus 0.1 mM phenylmethylsulfonyl fluoride (PMSF) and 1× proteinase
inhibitor mixture (Roche Molecular Biochemicals), broken by sonication, and centrifuged at 18,000 rpm for 1 h at 4 °C. The supernatant was aliquoted and stored at
80 °C. The amount of truncated AtTRP1 in each extract was estimated on 8% SDS-polyacrylamide gel; comparable amounts of truncated proteins were tested for DNA binding activity in
gel retardation experiments.
N-463 (Fig. 3A) was precipitated
with 45% (NH4)2SO4, and the pellet
was resuspended in 1× DNA-protein binding buffer plus 0.1 mM PMSF and 1× proteinase inhibitor mixture prior to the
fractionation on the plant telomeric sequence-specific DNA affinity
column. To purify the almost full-length protein of AtTRP1, the extract
containing the protein
N-12 (Fig. 3A) was fractionated
through the DEAE column in the equilibration buffer (20 mM
ethanolamine, pH 9.5, 1 mM dithiothreitol, 1 mM EDTA, 5% glycerol, 0.1 mM PMSF, and 1× proteinase
inhibitor mixture) containing stepwise increases in NaCl concentration.
Most of the degraded form of
N-12 appeared in both the flow-through
and the 0.1 M NaCl eluent. The intact form of
N-12 was
eluted from the DEAE column with 0.4 M NaCl and further
purified on the specific DNA affinity column in 1× DNA-protein binding
buffer containing stepwise increases in KCl concentration. Proteins
N-463 and
N-12 were eluted, respectively, at 0.6 and 1 M KCl from the DNA affinity column. The eluents containing
telomeric sequence binding activity were desalted, concentrated, and
purified once more on the same DNA affinity column, which was set up by
coupling the biotinylated duplex (TTTAGGG)8 DNA to the
streptavidin-agarose beads in 1× DNA-protein binding buffer. The
purified AtTRP1 derivatives were stored at
80 °C.
N-12
was either analyzed directly on the 8% denatured SDS-polyacrylamide
gel or subjected to gel retardation assay on the 4% native
polyacrylamide gel, electrophoretically transferred onto polyvinylidene
difluoride membranes, and hybridized with a polyclonal rabbit antiserum
against a synthetic peptide containing residues 34-45 of AtTRP1. The
membranes were washed to remove the unbound rabbit antiserum, incubated
with alkaline phosphatase-conjugated goat anti-rabbit IgG antibody,
and detected by chemiluminescent reaction using CSPD as substrate
(CLONTECH, CA).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Cloning of AtTRP1 and the
deduced primary structure of AtTRP1. A, restriction
maps of genomic and cDNA clones. Exons are indicated by solid
boxes. The TATA box and initiation and termination codons are
indicated. E, EcoRI; H,
HindIII; S, SalI; Xb,
XbaI; Xh, XhoI. B, the
deduced primary structure of AtTRP1. Clusters of basic residues are
underlined. Clusters of acidic residues are
underlined and shaded. Sequences rich in
glutamine are outlined. Residues in Myb domain are in
bold. C, homology between the sequences rich in
basic residues of AtTRP1 and the NLS of other nuclear proteins. R-NLS-M
and R-NLS-A represent, respectively, the NLS from the medial and
N-terminal regions of the maize R factor (47). D, alignment
of the Myb-like domain of AtTRP1 with those of the following proteins:
hTRF1 (27), hTRF2 (29), Taz1p (26), and Tbf1p (57). Amino acids in any
protein identical or similar to those in AtTRP1 are in
bold.
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Fig. 2.
Alignment among AtTRP1, RTBP1, and plant
initiator-binding proteins. Shaded regions indicate
residues identical or conserved between AtTRP1 and any one of the four
proteins.
N-463 retained the DNA binding activity (Fig. 3, A and C). This
truncated protein consists of the Myb motif and a C-terminal
polypeptide rich in glutamine (Fig. 1B). Removal of this
glutamine-rich polypeptide abolished the DNA binding activity of the
truncated protein
N-262, suggesting that the protein
N-463
contains a domain essential for recognizing the telomeric sequence.
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Fig. 3.
Mapping of the DNA-binding domain of
AtTRP1. A, schematic description of the DNA-binding
ability and the structure of AtTRP1 derivatives. The truncated proteins
N-12, 13-223 + 452-578,
N-262,
N-325,
N-393,
N-463,
and 263-522 are encoded, respectively, by the cDNAs
12,
11,
1,
2,
3,
4, and
31. The stronger the affinity between
the protein and the duplex (TTTAGGG)4 is indicated by
higher numbers with the symbol +. B, SDS-PAGE analysis.
C, gel retardation assay of bacterial extracts containing
truncated proteins is denoted at the top of each lane. A 6%
native polyacrylamide gel was used for gel retardation assay. The probe
was the duplex oligonucleotide (TTTAGGG)4. Lane
v is the extract from bacteria containing only the vector.
Lane m in B represents the molecular markers with
sizes of 94, 67, 43, 30, 20, and 14.4 kDa. Bands corresponding to the
truncated proteins in B are marked by asterisks.
Lane p in C represents the probe alone without
incubation with the bacterial extract.
N-12 is greater than that
of the protein
N-325 (Fig. 3, A and B), it is
expected that the complex containing
N-12 will migrate more slowly
on the native gel than the complex containing
N-325 in the reaction with an identical probe. However, incubation of the extract containing
N-12 with the probe (TTTAGGG)4 produced two weak
complexes that migrated faster than the single strong complex produced
by incubating the extract containing
N-325 with the identical probe
(Fig. 3C). The unexpected mobility of
N-12-containing
complexes may be due to the proteolysis of
N-12 in the complexes but
also could be due to some difference in the shapes between the
complexes containing
N-12 and that containing
N-325. Failure to
detect the strong slowly migrating complex(es) in either case indicates
that the probe (TTTAGGG)4 may be too short to be bound
efficiently by the protein
N-12, since the SDS-PAGE analysis
revealed that the amount of intact
N-12 is similar to that of intact
N-325 in the extract (Fig. 3B). A similar phenomenon was
also observed in the gel retardation assay of the extracts containing
the proteins 13-223 + 452-578 and
N-262 (Fig. 3C).
Based on the ability of binding the probe (TTTAGGG)4, these
truncated proteins with the functional DNA-binding domain can be
classified into two groups as follows: (a) group I,
including
N-12, 13-223 + 452-578 and
N-262, binding the probe poorly, and (b) group II, including
N-325,
N-393, and
N-463, binding the probe efficiently (Fig. 3A). The DNA
binding activities among the truncated proteins within each group
cannot be compared with one another until each one of them is purified.
N-463
containing the DNA-binding domain of AtTRP1 was purified to
near-homogeneity (Fig. 4A).
Seven duplex oligonucleotides, representing all possible combinations
of four contiguous and identical plant telomeric repeats with one
permutated nucleotide at a time, were used as probes for gel
retardation assay of the purified protein
N-463 (Fig.
4B). By using as little as 1 pmol of protein, one or two types of complexes (I and II) were observed, and as the protein concentration is increased, complexes with slower mobilities (III and
IV) were observed. In the reactions using the probe
(GGTTTAG)4, four complexes were observed, of which the
complex IV became predominant at high molar ratios of protein to DNA
probe (Fig. 4B, lanes 31-36), suggesting that
this protein recognizes each of the four sites in the probe
(GGTTTAG)4 with equal efficiency (Fig. 4C). The
probe (GGGTTTA)4 also formed four complexes with the same
protein, but the intensity of complex III was always higher than that
of complex IV, even when a high molar ratio of protein to the DNA probe
was used (Fig. 4B, lanes 25-30), suggesting that
one of the sites in the probe (GGGTTTA)4 is bound less
efficiently by this protein than the other three (Fig. 4C).
Similarly, one of the four sites in (TTTAGGG)4 is bound
less efficiently than the other three by this protein (Fig.
4B, lanes 1-6 and 4C). It should be
noticed that to form the complex IV, (TTTAGGG)4 requires
greater amounts of the protein
N-463 than does
(GGGTTTA)4 (Fig. 4B, lanes 1-6 and
25-30). In the reactions containing probes
(TAGGGTT)4 (Fig. 4B, lanes 13-18)
and (AGGGTTT)4 (Fig. 4B, lanes
19-24), complexes I-III were observed clearly but complex IV
only weakly. These data suggest that, in each probe, one site is
recognized poorly, and the other three sites are bound efficiently by
the protein
N-463. Only three complexes were formed in the reactions
containing probes (TTAGGGT)4 (Fig. 4B,
lanes 7-12) and (GTTTAGG)4 (Fig. 4B, lanes 37-42), suggesting that each probe consists of three
recognition sites for the protein
N-463. By combining these data, we
conclude that the isolated DNA-binding domain of AtTRP1 may recognize
each GGTTTAG repeat. Partial telomeric sequences such as TTTAG, GGTTTA, GGTTT, and GGTT may be also bound by this protein but less efficiently (Fig. 4C).
View larger version (79K):
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Fig. 4.
Identification of the core sequence in the
telomeric repeats recognized by the DNA-binding domain of AtTRP1.
A, SDS-PAGE (10%) analysis of the purified protein N-463
followed by staining with Coomassie Blue. B, gel retardation
assay of the purified protein
N-463. Increasing amounts of the
protein
N-463 were incubated with each of the indicated probes and
then analyzed on a 6% native polyacrylamide gel. Complexes
representing the probes bound by 1-4 molecules of
N-463 are denoted
to the left of the gel (roman numerals).
F represents the free probe. C, schematic
description of the possible binding sites for
N-463 along the length
of the oligonucleotides used in B. It is proposed, based on
the results shown in B, that each shaded block
represents a recognition site for the protein
N-463. The
darker the shaded block, the stronger the
affinity between the sequence in the block and the protein. Sequences
bound poorly by the protein
N-463 are italicized. Gaps
were introduced between the blocks for better visualization of the
boundary of each block.
N-12 was purified. SDS-PAGE
analysis of the purified
N-12 revealed that the resulting protein
N-12 appeared to be 90-95% pure and has an apparent molecular
mass of 83 kDa (Fig. 5A,
left panel). The discrepancy between the apparent and the
calculated molecular mass of AtTRP1 suggests that this protein migrates
anomalously on SDS gel. Since this protein was eluted from the DNA
affinity column and can be detected with the antibody against a peptide
near the N terminus of AtTRP1 (Fig. 5A, right panel), it has been suggested that this protein is probably the intact form of
N-12. This protein does not bind the duplex probe (TTTAGGG)4 (data not shown) and forms complexes only with
the probes carrying five or more GGTTTAG repeats (Fig. 5B),
suggesting that the minimum length of a telomeric DNA bound by the
protein
N-12 or AtTRP1 spans five GGTTTAG repeats.
N-12 binds
(GGTTTAG)8 more efficiently than (GGTTTAG)6,
whereas the probe (GGTTTAG)5 binds very little of
N-12
(Fig. 5B, lanes 6-20), indicating that AtTRP1
may require eight or more GGTTTAG repeats for optimal complex formation. Our data do not exclude the possibility that probes with
more than eight GGTTTAG repeats show an additional enhancement of
AtTRP1 binding.
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Fig. 5.
Determination of the minimum length of a
telomeric DNA bound by AtTRP1. A, SDS-PAGE analysis of
the purified protein N-12. The protein on the 8% denatured gel was
either stained with Coomassie Blue (left panel) or detected
with an antibody (diluted 1:1000) against a N-terminal peptide of
AtTRP1 by Western hybridization (right panel). B,
gel retardation assay. The reaction mixtures containing various amounts
of the protein
N-12 and each one of the denoted probes were analyzed
by electrophoresis on a 4% native polyacrylamide gel. C,
Western hybridization. The duplicate samples of those in lanes
6-10 of B were transferred to a membrane after gel
retardation assay and detected with antibody (diluted 1:100) as used in
A. Some of the complexes I and II are indicated by
asterisks for better visualization of the signals.
N-12, the
complexes formed with the probe (GGTTTAG)5 were transferred
from the gel to the membrane after gel retardation assay and were
detected with an antibody against a short peptide at the N terminus of AtTRP1 (Fig. 5C). The Western hybridization showed that both
complexes I and II reacted with the antibody, confirming that the
protein moiety in both complexes is
N-12.
N-12 binding between the
probe (GGTTTAG)8 and unlabeled duplex oligonucleotides
carrying plant telomere-related sequences and human telomeric repeats
was investigated (Fig. 6). Although both
oligonucleotides (GGTTTAG)4 and (GGTTTAG)8
consist of contiguous plant telomeric repeats, only the latter competes well for complex formation (Fig. 6, lanes 2-6), confirming
that the proteins
N-12 or AtTRP1 do not form any complex with the oligonucleotide (GGTTTAG)4 (Fig. 5B). The
sequences TTTTGGG and TTAAGGG have been found at subtelomeric or
telomeric regions in some plant genomes (6, 53, 54), but the
competition experiment showed that the oligonucleotides
(TTTTGGG)4 and (TTAAGGG)4 only compete weakly
in complex formation (Fig. 6, lanes 7-12). The oligonucleotide carrying the human telomeric repeat
(TTAGGG)4 is not an effective competitor (Fig. 6,
lanes 13-15). The single-stranded oligonucleotides (GGTTTAG)8 and (CTAAACC)8 fail
to compete in complex formation (data not shown).
View larger version (52K):
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Fig. 6.
Sequence specificity of AtTRP1.
The purified protein (0.2 µg) was allowed to react with 1 pmol of
duplex probe (GGTTTAG)8 in the presence of 20 and 40 (lanes 2 and 3) or 40, 80, and 160 (lanes
4-24) molar excess of the indicated duplex DNA as the
competitors. The reaction in lane 1 contained no competitor
DNA. The sequences for the binding sites of IBP1 and BPF1 are
respectively
G3AG3T3CTCTG3ACG3AGAG3AC
(51) and
A2GA2G2AGT2G2T2GAGA2T2A2
(50).
N-12 when they
were used as the probes (data not shown).
N-12 binds poorly to AtTRPro. Gel retardation assay also revealed
that no complex formed between the protein
N-12 and the probe
AtTRPro (data not shown). The combined results of Figs. 5 and
6 suggest that the protein AtTRP1 binds specifically to
telomeric DNA fragments containing at least five GGTTTAG repeats.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
N-12.
It is important to know whether the proteins HPPBF1 and AtTRP1 are
different classes of telomere-binding factors in A. thaliana, since they are highly homologous to each other in the
Myb-like DNA-binding domain (Fig. 2).
N-463,
N-393,
N-325,
N-262, and
N-12
(Figs. 3-5) indicates that the presence of the peptide containing
residues 13-325 in the N-terminal half of AtTRP1 may cause AtTRP1 to
recognize the longer telomeric DNA fragments. By using the longer
telomeric DNA fragments as the recognition sites not only makes the
binding specificity of AtTRP1 more stringent but also implies that this
protein may locate at the ends of plant chromosomes, which consist of
long stretches of telomeric repeats.
N-12 and the
telomeric DNA, the complex II migrates more slowly than the complex I
on the native gel (Fig. 5B), suggesting that the apparent
molecular weight of complex II is greater than that of complex I. This
led us to propose that the formation of complexes I and II may be attributed to the binding of one and two molecules of protein, respectively, to a single oligonucleotide. The complexes I appeared as
fuzzy and inconstant bands, suggesting that these complexes formed by
binding one protein to a single DNA molecule may be highly unstable.
Complex II is the major one formed in all of the reactions, suggesting
that AtTRP1 may bind plant telomeric DNA predominantly as a dimer. In
addition, complex II is much more stable than the complex I, raising
the possibility that two molecules of AtTRP1 may interact with each
other to form a dimeric protein that, perhaps, binds the DNA much more
tightly than does a single AtTRP1 molecule. Moreover, protein
N-12
forms complexes II with probes carrying various number of telomeric
repeats, implying that the protein AtTRP1 may have a flexible region
which allows the two DNA-binding domains of the putative dimeric
protein to recognize the telomeric DNAs with various lengths.
N-12 does not bind
the IBP1-binding site, which contains a single telomeric repeat (Fig.
6), suggesting that AtTRP1 does not recognize interstitial regions in
plant chromosomes that contain a single telomeric repeat. A survey has
shown that the 5' regions of some Arabidopsis genes contain
two or more non-contiguous telomeric repeats (39). Since the dimeric
hTRF1 can bind DNA fragments containing non-contiguous telomeric
repeats separated by non-telomeric sequences (55, 56), it would be of
interest to see whether the AtTRP1 protein would bind the
non-contiguous telomeric repeats at the 5' regions of these genes and
function as a transcriptional regulator. On the other hand, the
degraded AtTRP1 might play a role in transcriptional regulation of some
plant genes, since we have observed that the isolated DNA-binding
domain of AtTRP1 can recognize both the IBP1-binding site and the
AtTRPro DNA fragment (data not shown). It should be noticed that only
the C-terminal truncated proteins containing the Myb-like motif of IBP1
or BPF1 were shown to interact with their binding sites. It is not
clear whether the corresponding full-length proteins would recognize
the same binding site (50, 51).
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ACKNOWLEDGEMENTS |
---|
We thank C. C. Chen and G. D. Chang for a critical review of this manuscript.
![]() |
FOOTNOTES |
---|
* This work was supported by Grant NSC 88-2311-B-001-080 from the National Science Council and Academia Sinica in the Republic of China.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) ATH17722.
§ To whom correspondence should be addressed, Tel.: 886-2-27899590 (Ext. 216); Fax: 886-2-27827954; E-mail: bocmchen@ccvax.sinica.edu.tw.
Published, JBC Papers in Press, January 30, 2001, DOI 10.1074/jbc.M009659200
2 C. M. Chen, C. T. Wang, and C. H. Ho, unpublished data.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
kbp, kilobase pairs;
GAD, GAL4 activation domain;
RACE, rapid amplification of cDNA
ends;
TRPs, telomeric repeat-binding proteins;
PCR, polymerase chain
reaction;
bp, base pair;
PMSF, phenylmethylsulfonyl fluoride;
IPTG, isopropyl-1-thio--D-galactopyranoside;
PAGE, polyacrylamide gel electrophoresis;
NLS, nuclear localization
signals.
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