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RAV1, a novel DNA-binding protein, binds to bipartite recognition sequence through two distinct DNA-binding domains uniquely found in higher plants
Introduction
Materials And Methods
Plasmid construction
Expression of recombinant proteins
Electrophoretic mobility shift assays (EMSAs)
BSSAs
Methylation interference experiments
Results
Isolation of cDNA clones for RAV1 and RAV2
RAV proteins contain an AP2-like domain
Expression of RAV1
RAV1 specifically binds to DNA with bipartite sequence motifs
RAV1 interacts with the CAACA and CACCTG motifs
RAV1 binds to DNA as a monomer
The AP2 and B3-like domains of RAV1 possess DNA-binding activity
The AP2 and B3-like domains recognize the CAACA and CACCTG motifs, respectively
Discussion
Acknowledgements
References
RAV1, a novel DNA-binding protein, binds to bipartite recognition sequence through two distinct DNA-binding domains uniquely found in higher plants
DDBJ/EMBL/GenBank accession nos AB013886 and AB013887
ABSTRACT
INTRODUCTION
To date, an increasing number of sequence-specific DNA-binding proteins or transcription factors have been cloned from higher plants using either genetic or biochemical approaches. Most of these plant proteins fall into families of transcription factors widely distributed in eukaryotes (1). The members of each family share evolutionarily conserved DNA-binding motifs, such as zinc finger, basic-leucine zipper (bZIP), basic-helix-loop-helix (bHLH), MADS box, homeo-box, etc. A few families of transcription factors such as AP2 domain protein family (2,3) and the family that comprises the product of the Viviparous1 locus (VP1) of maize (4) and its orthologues such as Arabidopsis ABI3 (5) have been found only in higher plants.
The AP2 domain was first identified as a DNA-binding domain conserved in a family of tobacco ethylene response element binding proteins (EREBPs) (6), and later found to be conserved in Arabidopsis APETALA2 (AP2) (7), which is involved in flower development. The number of different proteins containing an AP2-like-domain in a plant species appears to be strikingly large. We have identified >30 Arabidopsis ESTs encoding different AP2-like-domain-containing proteins. The number of genetically and/or biochemically characterized AP2-like-domain-containing proteins is also increasing. These include Arabidopsis AINTEGUMENTA (8,9), TINY (10), CBF1 (11) and ABI4 (12).
VP1 controls the expression of abscisic acid (ABA)-regulated genes such as Em (4) and C1 (13), which are associated with seed maturation (14). VP1/ABI3 factors have four amino acid sequence domains, namely A1 domain in the acidic N-terminal region and three basic regions designated B1, B2 and B3, which are conserved among plant species (15,16). The B3 domain lies in the C-terminal region and comprises the largest contiguous block of amino acid identity among the VP1/ABI3 homologues/orthologues from various plant species (5,17,18). Previously, it had not been possible to detect sequence-specific DNA-binding activity with recombinant VP1 protein. However, Suzuki et al. (16) have recently reported that the B3 domain has a cryptic DNA-binding activity specific to the Sph element, which is required for the regulation of the C1 promoter by VP1. More recently, ARF1 (auxin response element binding factor 1) (19) and FUSCA3 (FUS3) (20) have been shown to contain a B3-like DNA-binding domain. Furthermore, we have identified other B3-like domain-containing proteins, by surveying the Arabidopsis EST and genome databases.
In the present study, we have characterized full-length cDNAs corresponding to Arabidopsis ESTs (21) that encode B3-like domain-containing proteins. We designated these proteins RAV1 and RAV2 (RAV: for Related to ABI3/VP1). We initiated this study to elucidate the biochemical function of the highly conserved B3 domain of VP1/ABI3 because nothing was known. As mentioned above, however, the DNA-binding activity of the B3 domain of VP1 was reported during the course of this study (16). Although we also have independently found in this study that the B3-like domain of RAV1 has a sequence-specific DNA-binding activity, characterization of RAV1 and RAV2 cDNAs has led us to another unexpected finding that both proteins have an AP2-like domain in addition to the B3-like domain. This finding prompted us to further characterize the DNA-binding properties of RAV1 protein because no transcription factors with two or more DNA- binding domains of distinct types have been previously identified in higher plants. Even in other eukaryotes, only a few families of transcription factors with multiple distinct DNA-binding domains are known. These include POU-domain proteins (22) and Pax proteins (23), both of which have been studied extensively with respect to how the multiple DNA binding structures contribute to the specificity and affinity of DNA-binding (24,25). The POU-domain consists of two subdomains, a POU-specific domain (POUS) and a POU-type homeo-domain (POUHD), each of which can bind to DNA. The paired-domain (PD) of Pax proteins also consists of two subdomains, PAI and RED, both of which contain a helix-turn-helix (HTH) DNA-binding motif. Many Pax proteins contain a homeo-domain (HD) in addition to PD. Thus, these proteins contain three HTHs and have been shown to recognize different types of target sites using multiple combinations of their HTHs (24).
We demonstrate here that the two DNA-binding domains of RAV1 can separately recognize each of two motifs that constitute a bipartite binding sequence and together cooperatively enhance its DNA-binding affinity and specificity.
MATERIALS AND METHODS
Plasmid construction
Plasmids for production of glutathione S-transferase (GST) fusion proteins with a fragment of RAV1 containing amino acid residues 8-344 (GRAV1-ent), 8-288 (GRAV1-dC1), 8-200 (GRAV1-dC2), 8-169 (GRAV1-dC3), 8-73 (GRAV1-dC4) or 118-344 (GRAV1-dN) were constructed using an expression vector pGEX-5X-3 (Pharmacia) by standard recombinant DNA techniques including polymerase chain reaction (PCR) (26) . The sequences of 5[prime] junctions between the vector and RAV1 sequence of the constructed plasmids were: 5[prime]-GGTCTGGGATCCATGAGAGTACTACAAGT-3[prime] (GRAV1-ent, GRAV1-dC1, GRAV1-dC2, GRAV1-dC3 and GRAV1-dC4) ; 5[prime]-CCCAGGAATTCTCATTCGAAATCT-3[prime] (GRAV1-dN), where the sequences of vector portions are italicized. The sequences of the 3[prime] junctions were: 5[prime]-AAAAAAAAAAACTCGAGCGGCCGC-3[prime] (GRAV1-ent and GRAV1-dN); 5[prime]-TCTAACGGTCAGGGGGGGGCCCGGGGTCGACTCGAGC-3[prime] (GRAV1-dC1); 5[prime]-TGGGAAGCTAAGGGGGGGCCCGGGGTCGACTCGAGC-3[prime] (GRAV1-dC2); 5[prime]-AGGACGTTGTTGGGTCGACTCGAGC-3[prime] (GRAV1-dC3); 5[prime]-GGAAGATGGGGGGGTCGACTCGAGC-3[prime] (GRAV1-dC4).
To construct a plasmid for production of poly-histidine-tagged RAV1 (HRAV1), the entire RAV1 cDNA fragment was inserted into the blunt-ended XhoI digest of pET16b vector (Novagen). The sequences of 5[prime] and 3[prime] junctions were 5[prime]-CATATGCTCGAGATCCATGAGAGTACTACAAGT-3[prime] and 5[prime]-AAAAAAAAAAACTCGATCGAGGATCCGGCTG-3[prime], respectively.
Expression of recombinant proteins
For the expression of recombinant proteins, the JM109 Escherichia coli host strain was transformed with the plasmids described above. Purification of the recombinant proteins by affinity chromatography using glutathione-Sepharose (Pharmacia) or nickel ion resin (Novagen) was performed by the procedures recommended by the manufacturers. The purified recombinant proteins were analyzed for purity and integrity by SDS-PAGE (27). The concentration of each protein solution was determined by the method of Bradford (28).
Electrophoretic mobility shift assays (EMSAs)
Standard binding reaction in a total volume of 20 µl was performed by incubating an appropriate amount of purified recombinant protein with 10 fmol of 32P-labeled probe DNA and 2 µg of poly(dI-dC).poly(dI-dC) in B buffer [25 mM HEPES-KOH, pH 7.5, 100 mM KCl, 0.1 mM EDTA, 10% (v/v) glycerol, 1 mM dithiothreitol] at room temperature for 20 min. The binding reactions were resolved on a 4% polyacrylamide gel run in0.25× TBE buffer (22.5 mM Tris-borate, pH 8.0, 0.25 mM EDTA). To prepare the probes used in the experiments described in Figure
BSSAs
BSSAs were performed essentially as described by Gogos et al. (29). Binding site sequences were selected from 30 bp random sequences, which were flanked by primer sequences of 20 bp at both ends, [5[prime]-AACGGTACCAGAAGCTTACC(N)30CCAGAATTCGAGCTCTTCGT-3[prime]]. The binding reaction contained 500 ng (for the first selection cycle) or 200 ng (for the second selection cycle and thereafter) of the 32P-labeled DNA, 500 ng (for the first selection cycle) or 100 ng (for the second selection cycle and thereafter) of purified GRAV1-ent protein and 1 µg (for the first selection cycle) or 2 µg (for the second selection cycle and thereafter) of poly(dI-dC).poly(dI-dC) in B buffer at a total volume of 50 µl (for the first selection cycle) or 20 µl (for the second selection cycle and thereafter). The conditions for the PCR-amplification of selected sequences were: 17 cycles of consecutive reactions at 94°C (30 s), 55°C (30 s) and 72°C (45 s). After the fourth and sixth rounds of selection, PCR-amplified double-stranded DNA fragments (70 bp) were digested with SacI and KpnI, and cloned into pBluescript. These pBluescript plasmid clones were sequenced.
Methylation interference experiments
Plasmid DNAs containing the sequences selected by BSSAs were digested with SacI and KpnI, and blunt-ended with T4 DNA polymerase. The resultant 52 bp DNA fragments were cloned again into EcoRV site of pBluescript. The inserts of these plasmid clones were cut out with XhoI or XbaI, labeled by the fill-in reaction, and then digested with either XbaI or XhoI, which was not used for the first digestion. The resultant 119 bp XbaI/XhoI fragments were used for methylation interference experiments (26). The binding reactions and separation of protein-bound DNAs were performed as described above for EMSAs.
RESULTS
Isolation of cDNA clones for RAV1 and RAV2
The amino acid sequence at the C-terminal region (amino acid residues 507-620) of maize VP1 that contains the highly conserved B3 domain was used for a similarity search against GenBank's EST database (dbEST) by the tBLSTn algorithm. The search revealed the presence of at least two types of Arabidopsis cDNAs potentially encoding amino acid sequences significantly similar to the B3 domains of maize VP1 and its homologues from other plant species. Two representative Arabidopsis EST clones, 117P5T7 and 97E4T7 (GenBank accession nos T43470 and T21748, respectively), which had been obtained from the Arabidopsis Biological Resource Center (ABRC; Ohio State University, OH), were used to screen a cDNA library prepared from 10 day old seedlings of Arabidopsis thaliana (ecotype Columbia). Among 13 and 15 positive clones obtained by hybridization screening with 117P5T and 97E4T7, respectively, those with the longest cDNA inserts were chosen for sequence analyses. These cDNA clones, pRAV1-01 corresponding to 117P5T7 and pRAV2-01 corresponding to 97E4T7, had cDNA inserts of 1276 and 1315 bp, which encoded proteins of 344 and 352 amino acids, respectively. We designated pRAV1-01 and pRAV2-01-encoded proteins RAV1 and RAV2, respectively. The deduced amino acid sequences of RAV1 and RAV2 are compared in Figure Figure 1. Amino acid sequences of RAV1 and RAV2. Amino acid residues identical to those of RAV1 are indicated by dots (···). Gaps are indicated by dashes (-). (A) Amino acid sequences of RAV1 and RAV2 deduced from the nucleotide sequences of the cDNA clones, pRAV1-01 and pRAV2-01, are compared. The N-terminal and C-terminal conserved sequence blocks are indicated by lines. (B) Amino acid sequence comparison of the C-terminal portion of RAV1 and RAV2 to those of B3 or B3-like domains present in maize VP1, and Arabidopsis ABI3 and ARF1. Amino acid residue numbers of the regions compared are indicated in parentheses. (C) Amino acid sequences of part of the N-terminal region of RAV1 and RAV2 are compared to those of AP2 domains found in Arabidopsis APETALA2 and tobacco EREBP1. Amino acid residue numbers of the regions compared are indicated in parentheses. (D) Schematic illustrations for domain organizations of RAV1/RAV2, APETALA2, EREBPs and VP1/ABI3. Sequence similarity searches against databases revealed a striking feature of the RAV proteins. The N-terminal conserved block of RAV1 and RAV2 (from amino acid residues 58 to 120 for RAV1 and from 61 to 123 for RAV2) was found to contain a region that exhibited significant homology to the AP2 domains identified in various plant proteins, including Arabidopsis APETALA2 (7) and tobacco EREBPs (Fig. Northern hybridization analysis (Fig. Figure 2. Northern blot hybridization analysis of RAV1 mRNA. Ten micrograms of total RNAs prepared from indicated organs of Arabidopsis plants were subjected to northern blot hybridization analysis using RAV1 cDNA as a probe. The photograph of ethidium bromide-stained agarose gel before RNA transfer is shown at the bottom. As RAV proteins possessed a putative DNA binding domain (AP2-like domain), we attempted to confirm their sequence-specific DNA-binding activity, and to determine their recognition sequences by performing BSSAs. A GST fusion protein (GRAV1-ent) that contained almost the entire RAV1 coding sequence (from amino acid residues 8 to 344) was produced in bacteria and purified. Using this fusion protein, recognition sequences of RAV1 were selected from random 30mers flanked by primer sequences, by repeating cycles of EMSAs and PCR amplification. The abundance of DNA sequences recognized by GRAV1-ent increased as the selection cycle proceeded, while GST alone did not select any specific sequences (data not shown). Oligonucleotides recovered after the fourth and sixth rounds of selection were PCR-amplified and cloned into a plasmid vector. Each of those sequences was tested for binding to GRAV1-ent by EMSA. DNA sequences were analyzed for 68 independent clones (19 from the fourth round and 49 from the sixth round) among those whose binding to GRAV1-ent were confirmed (Fig. Figure 3. Nucleotide sequences selected by BSSAs. (A) Sixty-eight nucleotide sequences selected after the fourth or sixth round of BSSAs are classified according to the type and combination of sequence motifs. The number of sequences in each category is indicated in parentheses. The CAACA and CACCTG (either in the forward or reverse orientation) motifs are italicized/shaded and just shaded, respectively. (B) Consensus sequences of CAACA and CACCTG motifs deduced from the frequencies of base-occurrence at each position. The numbers of sequences with the indicated bases at each position are shown. Bases corresponding to the linker sequences, indicated with lower case letters, are excluded from counting. Figure 4. RAV1 interacts with both the CAACA and the CACCTG motifs. Methylation interference experiments were conducted using sequences 6-1, 6-2, 4-7 and 4-18 (Fig. 3), and GRAV1-ent. Results with only the bottom strand (6-1) or both strands (6-2, 4-7 and 4-18) are shown. `F' and `B' indicate free and protein-bound DNA, respectively. Bands corresponding to G residues that strongly and less strongly interfered with protein binding when methylated are indicated by closed and open circles, respectively. Results are summarized at the bottom. Note that in sequence 4-18, the CACCTG motif is in the reverse orientation relative to the CAACA motif. In summary, RAV1 binds specifically to bipartite sequences comprising two unrelated motifs, separated by various spacings, and in different relative orientations. Among the analyzed 68 sequences, there were three sequences that did not follow this rule. Interestingly, one of these sequences (4-17) did not contain a CACCTG motif but was instead composed of two CAACA motifs (Fig. To confirm that RAV1 actually interacts with both CAACA and CACCTG motifs, methylation interference experiments were conducted using four representative selected sequences (sequence 6-1, 6-2, 4-7 and 4-18; Fig. We tested whether or not RAV1 binds to a target sequence as a dimer or other oligomer by mixing recombinant RAV1 proteins fused to tags of different sizes. As shown in Figure To localize regions of RAV1 responsible for sequence-specific DNA-binding, various deletion mutants of GST-RAV1 fusion protein (Fig. GRAV1-ent (ent) bound equally well to the tested sequences except for sequences 6-31 and 4-17. In contrast, the autonomous DNA-binding activities of mutant proteins containing either the AP2 or B3-like domain alone exhibited a strong dependence on the target sequences (Fig. GRAV1-dC3 hardly bound (sequences 4-3, 6-25, 4-7 and 6-31) or only weakly with different efficiencies (sequences 6-2, 6-1 and 4-18) to the sequences containing a single copy of the CAACA motif, although the efficiency of the binding to sequence 6-2, which exhibited the strongest binding among them, was very low compared with that to sequence 4-17. Such differences could not simply be accounted for by the deviation from the consensus sequence of the CAACA motif (Fig. Figure 5. RAV1 binds to DNA as a monomer. Designation of each fusion protein is shortened by omitting the suffix `ent'. The probe used in the EMSAs was sequence 6-1 (Fig. 3). (A) EMSAs using GST (GRAV1-ent) or poly-histidine-tag (HRAV1-ent) fusion protein, or a mixture of them, which had been incubated for 16 h. (B) EMSAs using GRAV1-ent protein from which GST tag was partially removed. Twenty nanograms of GRAV1-ent was digested with the indicated amount of factor Xa for 1 h at 25°C. The digestion reactions were terminated by the addition of phenylmethanesulfonyl fluoride, and the digests were used for EMSAs. Near-complete digestion was obtained with 0.8 ng of factor Xa. Note that the mobility of the complex with RAV1 without the GST tag is approximately the same as that of the HRAV1-ent complex because the molecular masses of the two proteins are very similar to each other. Figure 6. The B3 and AP2-like domains have autonomous DNA-binding activity. EMSAs were performed using GST fusion protein of RAV1 and its deletion derivatives. Designation of each fusion protein is shortened by omitting the prefix `GRAV1' in (B) and (C), and also in Figure 7. (A) Schematic illustrations of the GST fusion proteins used in the experiments. The regions of RAV1 protein fused to GST are indicated by amino acid residue numbers on the right. Filled and hatched boxes indicate the AP2 and B3-like domains, respectively. (B) EMSAs using the GST-RAV1 fusion protein and its deletion derivatives shown in (A) at a low (50 ng) or high (500 ng) protein concentration. The probe used was sequence 6-1 [Fig. 3 and (C) in this Figure]. (C) EMSAs using GST fusion proteins with the entire region (GRAV1-ent), the B3-like domain alone (GRAV1-dN) or the AP2-like domain alone (GRAV1-dC3). The amount of protein used was 50 ng for GRAV1-ent or 500 ng for GRAV1-dN and GRAV1-dC3. Several different sequences selected by BSSAs (Fig. 3) were used for probes as indicated. The binding properties of the isolated AP2 and B3-like domains to various target sequences suggested that the two domains separately recognize each of the two motifs in the bipartite binding sequences (Fig. Figure 7. The AP2 and B3-like domains of RAV1 separately recognize the CAACA and CACCTG motifs, respectively. (A) EMSAs with a wild-type and mutant recognition sequences. Wild-type (WT) duplicated sequence 6-1 (2× 6-1) and its mutants, in which base-changes were introduced in either the CAACA (mACA) or CACCTG (mCCT), or in both (mACA/CCT), were used for probes as indicated. Bases changed from the wild-type sequence are indicated with lower case letters. The GST fusion protein (Fig. 6A) and amount of protein used are indicated at the top of each panel. The band indicated by an arrow probably represents a complex, in which both of the two binding sites are occupied by GRAV1-ent molecules. The bands indicated by asterisks were resulted from contaminated bacterial protein(s). This conclusion is based on the observation that different protein preparations gave variable relative strength of these bands. (B and C) Methylation interference experiments using GRAV1-dN (B) and GRAV1-dC3 (C). Probes used were as indicated. We have cloned and characterized Arabidopsis cDNA clones for RAV1 and RAV2, which were shown to contain AP2 and B3-like domains. Both AP2 and B3-like domains of RAV1 were demonstrated to possess autonomous DNA-binding activities specific for CAACA and CACCTG motifs, respectively, which together constitute a bipartite high-affinity binding site for RAV1. The B3 domain was originally identified as a highly conserved region found near the C-terminus of VP1/ABI3 proteins from various plant species. Owing to the large scale sequence analyses of Arabidopsis cDNAs (21), we were able to recognize the existence of B3-like-domain-containing proteins other than VP1/ABI3. Despite the relatively weak conservation between the B3-like domains of RAV proteins and those of VP1/ABI3, the commonly shared DNA-binding function further establishes their evolutionary relatedness. ARF1 (19) and FUS3 (20) also contain a B3-like domain, and the DNA-binding activity of ARF1 was mapped to a region containing the B3-like domain as well (19). The amino acid sequence conservation between the B3-like domains of ARF1 and the B3 domains of VP1/ABI3 is weaker than that between RAV and VP1/ABI3 proteins (Fig. In addition to the B3-like domain, RAV1 and RAV2 contain an AP2-like domain, another DNA binding domain also identified uniquely in higher plants. Some AP2-domain-containing proteins such as APETALA2 (7) and AINTEGUMENTA (8), contain two AP2 domains in the same molecule. Thus, these proteins appear analogous to RAV proteins in that they contain two DNA-binding domains. A single AP2 domain in these proteins may not be sufficient to achieve a required binding affinity and/or specificity as demonstrated for RAV1 in this study. While a larger amount of protein and/or multiple recognition sites were required to detect DNA-binding of fusion proteins with either the AP2 or B3-like domain alone, the entire RAV1 protein showed a strong binding to sequences with a single bipartite recognition site. The EMSAs and methylation interference experiments described in Figure GRAV1-ent bound to sequence 6-31 and 4-17 with relatively low efficiencies compared to other sequences. The weak binding to 4-17 is considered to be due to its lack of a CACCTG motif. The reason for the relatively weak binding to sequence 6-31 is not clear. It is difficult to explain it by the differences in sequence on the basis of on current data. The CACCTG motif of sequence 6-31 is located closely to the end of the probe oligonucleotide, whereas those of other sequences are not. Thus, the relative position of recognition site in a probe DNA might affect the efficiency of binding. One of the remarkable features of RAV1 is its flexible binding to the bipartite recognition sequences with the two different motifs being variably spaced and oriented. A glycine-rich region (amino acid residues 158-180) and a region rich in charged amino acids and poor in hydrophobic amino acids (amino acid residues 149-165) exist between the AP2 and B3-like domains. These regions may contribute to the flexibility. The flexible properties of binding might have served to expand or create the repertoire of target sites according to necessity during evolution. In other words, RAV1 might have evolved to regulate a wider range of genes rather than a more specific set of genes. It has been shown that the POUS and POUHD subdomains of the POU domain in Oct-1, each having a weak but autonomous DNA-binding activity, cooperatively elaborate a high affinity binding. Similarly, HD and the PAI subdomain of PD in Pax family proteins can independently bind to DNA but together show an intramolecular cooperativity when they bind to a certain type of target sites (24). In addition, flexible positioning of two DNA-binding domains to variable bipartite recognition sequences has been also reported for POU-domain proteins (32,33). Like these proteins, the two DNA-binding domains of RAV1 cooperatively function to achieve a high affinity binding. However, it should be noted that RAV1 and RAV2 are the first examples of the higher plant proteins containing two distinct DNA-binding domains and that both of the DNA-binding domains are uniquely found in higher plants. Although neither their physiological functions nor target genes are currently known, RAV proteins are likely to be involved in some biological processes specially evolved in higher plants. The entire genome sequence of Arabidopsis, which will be available in a few years, would help to identify the target genes. Using the PatMatch program at the AtDB (http://genome-www2.stanford.edu/cgi-bin/AtDB/PATMATCH/nph-patmatch ), we searched for potential RAV1 binding sequences in the current Arabidopsis sequence databases. By this search, we have identified~450 sequences that match a sequence pattern C (A/C/G)ACA-(N)3-9ACCTG(A/G), which mostly covers the RAV1 binding sequences identified by BSSAs. Most of these sequences are found in cDNAs, exons and introns of putative genes and unidentified regions of the genome. But some sequences are found in the promoter regions of known genes. These include the BAS1 (34) and ABP (35) genes, which encode a 2-Cys peroxiredoxin and auxin-binding protein, respectively. We confirmed by EMSAs that RAV1 can actually bind to these sequences with efficiencies comparable to that for sequence 6-1 (Y.Kagaya and T.Hattori, unpublished results). Although further study is necessary to confirm whether RAV1 is actually involved in the regulation of these genes, we have recently observed increased levels of BAS1 expression in transgenic Arabidopsis lines that overexpress RAV1 (Y.Kagaya and T.Hattori, unpublished results). Thus, such a strategy to identify the target genes appears to be promising. Once target genes are identified, the information obtained in the present study will contribute to the understanding of the regulatory mechanism of their transcription. We wish to thank Dr Mauricio Bustos (University of Maryland at Baltimore County) for critical reading of the manuscript. This work was supported in part by Grant-in-Aids for Scientific Research on Priority Areas (`Molecular Basis of Flexible Organ Plans in Plant', No. 06278102) to T.H. and for JSPS Fellows (No. 8878) to Y.K. from the Ministry of Education, Science, Sports and Culture, Japan, and by a project grant from the Ministry of Agriculture, Forestries and Fisheries, Japan to T.H. Y.K. is a JSPS Research Fellow.
RAV proteins contain an AP2-like domain
Expression of RAV1
RAV1 specifically binds to DNA with bipartite sequence motifs
RAV1 interacts with the CAACA and CACCTG motifs
RAV1 binds to DNA as a monomer
The AP2 and B3-like domains of RAV1 possess DNA-binding activity
The AP2 and B3-like domains recognize the CAACA and CACCTG motifs, respectively
DISCUSSION
ACKNOWLEDGEMENTS
REFERENCES
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