DNA Binding Experiments Research Articles

Identification of a novel 70 kDa protein that binds to the core promoter element and is essential for ribosomal DNA transcription.

Mammalian ribosomal RNA genes (rDNA) are transcribed by RNA polymerase I and at least two auxiliary factors, UBF and SL1/TFID/TIF-IB. It has also been reported that an additional factor(s) is required to reconstitute efficient initiation of rDNA transcription in vitro, depending upon the procedures of chromatographic separation. In an attempt to elucidate the molecular identity of such yet uncertain activities, we have developed agarose gel shift and UV cross-linking assays to detect proteins directly bound to the core promoter region of murine rDNA. With these techniques, we identified a 70 kDa protein (p70) in the flow-through fraction of a phosphocellulose column (TFIA-fraction). Interestingly, the binding of p70 to the rDNA core promoter was observed only in the presence of the SL1-containing fraction. The probable human orthologue of p70 was also detected in HeLa cells. Consistent with the observation that p70 bound to the core promoter only in the presence of the TFIA- and SL1-fractions, alteration of DNase I footprint pattern over the core promoter element was demonstrated by cooperative action of the TFIA- and SL1-fractions. A reconstituted in vitro transcription assay with further purified p70 indicated that p70 was required for accurate initiation of rDNA transcription. These results indicate that the p70 identified recently by the current DNA-binding experiments represents a novel transcription factor in rDNA transcription.

Involvement of STAT5 (signal transducer and activator of transcription 5) and HNF-4 (hepatocyte nuclear factor 4) in the transcriptional control of the hnf6 gene by growth hormone.

HNF-6 is a tissue-restricted transcription factor that participates in the regulation of several genes in liver. We reported earlier that in adult rats, HNF-6 mRNA concentration in liver drops to almost undetectable levels after hypophysectomy and returns to normal after 1 week of GH treatment. We now show that this results from a rapid effect of GH, and we characterize its molecular mechanism. In hypophysectomized rats, HNF-6 mRNAs increased within 1 h after a single injection of GH. The same GH-dependent induction was reproduced on isolated hepatocytes. To determine whether GH regulates hnf6 expression at the gene level, we studied its promoter. DNA binding experiments showed that 1) the transcription factors STAT5 (signal transducer and activator of transcription 5) and HNF-4 (hepatocyte nuclear factor 4) bind to sites located around -110 and -650, respectively; and 2) STAT5 binding is induced and HNF-4 binding affinity is increased in liver within 1 h after GH injection to hypophysectomized rats. Using transfection experiments and site-directed mutagenesis, we found that STAT5 and HNF-4 stimulated transcription of an hnf6 gene promoter-reporter construct. Furthermore, GH stimulated transcription of this construct in cells that express GH receptors. Consistent with our earlier finding that HNF-6 stimulates the hnf4 and hnf3beta gene promoters, GH treatment of hypophysectomized rats increased the liver concentration of HNF-4 and HNF-3beta mRNAs. Together, these data demonstrate that GH stimulates transcription of the hnf6 gene by a mechanism involving STAT5 and HNF-4. They show that HNF-6 participates not only as an effector, but also as a target, to the regulatory network of liver transcription factors, and that several members of this network are GH regulated.

An engineered <i>lox</i> sequence containing part of a long terminal repeat of HIV-1 permits Cre recombinase-mediated DNA excision

In our previous report, one 34-bp sequence from a long terminal repeat (LTR) of human immunodeficiency virus type 1 (HIV-1) clone, loxLTR-1, was proposed as a target site for site-specific excision by modified Cre recombinase. To support this suggestion, an engineered lox sequence, designated loxIL1, was made. This variant lox has the corresponding sequence of loxLTR-1 at the spacer region and the last two bases of inverted repeat sequence. Through in vitro recombination assay, loxIL1 also allowed the wild-type Cre to specifically recombine the sequence. An in vitro DNA binding experiment with mutants CreK244R and CreK244L revealed that lysine 244 of Cre plays an important role in interaction with the engineered lox. This result suggests that loxLTR-1 would be a candidate for antiviral strategy using site-specific recombinase.

A variant form of the nuclear triiodothyronine receptor c-ErbAalpha1 plays a direct role in regulation of mitochondrial RNA synthesis.

In earlier research, we identified a 43-kDa c-ErbAalpha1 protein (p43) in the mitochondrial matrix of rat liver. In the present work, binding experiments indicate that p43 displays an affinity for triiodothyronine (T3) similar to that of the T3 nuclear receptor. Using in organello import experiments, we found that p43 is targeted to the organelle by an unusual process similar to that previously reported for MTF1, a yeast mitochondrial transcription factor. DNA-binding experiments demonstrated that p43 specifically binds to four mitochondrial DNA sequences with a high similarity to nuclear T3 response elements (mt-T3REs). Using in organello transcription experiments, we observed that p43 increases the levels of both precursor and mature mitochondrial transcripts and the ratio of mRNA to rRNA in a T3-dependent manner. These events lead to stimulation of mitochondrial protein synthesis. In transient-transfection assays with reporter genes driven by the mitochondrial D loop or two mt-T3REs located in the D loop, p43 stimulated reporter gene activity only in the presence of T3. All these effects were abolished by deletion of the DNA-binding domain of p43. Finally, p43 overexpression in QM7 cells increased the levels of mitochondrial mRNAs, thus indicating that the in organello influence of p43 was physiologically relevant. These data reveal a novel hormonal pathway functioning within the mitochondrion, involving a truncated form of a nuclear receptor acting as a potent mitochondrial T3-dependent transcription factor.

Cytotoxicity, Apoptosis, and in Vitro DNA Damage Induced by Potassium Chromate

Cr6+ is a known human cytotoxic and carcinogenic agent that requires intracellular reduction for activation. We have analyzed the cytotoxic and DNA binding properties of K2CrO4 (Cr6+) in comparison with those of Cl3Cr (Cr3+). The results indicate that K2CrO4 exhibits higher cytotoxicity than Cl3Cr in several human and murine cell lines. The cytotoxic activity of K2CrO4 is also indicated by the fact that is able to produce cell killing through apoptosis in cisplatin-resistant cells transformed by H-ras oncogene. Moreover, in vitro DNA binding experiments show that, in the presence of ascorbate (the major intracellular reductant of Cr6+), K2CrO4 induces both interstrand cross-links and strand breaks. Because the chromate anion is by itself unreactive toward DNA, these data suggest that the cytotoxicity of K2CrO4 may be associated with the DNA binding of reactive intermediate chromium species resulting from reduction of Cr6+.

Ternary complex formation between the MADS-box proteins SQUAMOSA, DEFICIENS and GLOBOSA is involved in the control of floral architecture in Antirrhinum majus

In Antirrhinum, floral meristems are established by meristem identity genes. Floral meristems give rise to floral organs in whorls, with their identity established by combinatorial activities of organ identity genes. Double mutants of the floral meristem identity gene SQUAMOSA and organ identity genes DEFICIENS or GLOBOSA produce flowers in which whorled patterning is partially lost. In yeast, SQUA, DEF and GLO proteins form ternary complexes via their C-termini, which in gel-shift assays show increased DNA binding to CArG motifs compared with DEF/GLO heterodimers or SQUA/SQUA homodimers. Formation of ternary complexes by plant MADS-box factors increases the complexity of their regulatory functions and might be the molecular basis for establishment of whorled phyllotaxis and combinatorial interactions of floral organ identity genes.

Interactions of mammalian proteins with cisplatin-damaged DNA

We have undertaken the systematic isolation and characterization of mammalian proteins which display an affinity for cisplatin-damaged DNA. Fractionation of human cell extracts has led to the identification of two classes of proteins. The first includes proteins that bind duplex DNA in the absence of cisplatin damage and retain their affinity for DNA in the presence of cisplatin–DNA adducts. The DNA-dependent protein kinase (DNA-PK) falls into this class. The inhibition of DNA-PK phosphorylation activity by cisplatin-damaged DNA has led to the hypothesis that cisplatin sensitization of mammalian cells to ionizing radiation may be mediated by DNA-PK. The second class of proteins identified are those which display a high relative affinity for cisplatin-damaged DNA and a low affinity for undamaged duplex DNA. Proteins that fall into this class include high mobility group 1 protein (HMG-1), replication protein A (RPA) and xeroderma pigmentosum group A protein (XPA). Each protein has been isolated and purified in the lab. The interaction of each protein with cisplatin-damaged DNA has been assessed in electrophoretic mobility shift assays. A series of DNA binding experiments suggests that RPA binds duplex DNA via denaturation and subsequent preferential binding to the undamaged DNA strand of the partial duplex. DNA substrates prepared with photo-reactive base analogs on either the damaged or undamaged DNA strand have also been employed to investigate the mechanism and specific protein–DNA interactions that occur as each protein binds to cisplatin-damaged DNA. Results suggest both damage and strand specificity for RPA and XPA binding cisplatin-damaged DNA.

RheA, the repressor of hsp18 in Streptomyces albus G.

In Streptomyces albus, Hsp18, a protein belonging to the family of small heat-shock proteins, can be detected only at high temperature. Disruption of orfY, located upstream and in the opposite orientation to hsp18, resulted in an elevated level of hsp18 mRNA at low temperature. Genetic and biochemical experiments indicated that the product of orfY, now called RheA (Repressor of hsp eighteen), directly represses hsp18. In Escherichia coli, an hsp18'-bgaB transcriptional fusion was repressed in a strain expressing S. albus RheA. DNA-binding experiments with crude extracts of E. coli overproducing RheA indicated that RheA interacts specifically with the hsp18 promoter. Transcription analysis of rheA in the S. albus wild-type and in rheA mutant strains suggested that RheA represses transcription not only of hsp18 but also of rheA itself.

Careers-perspective interview

I have a PhD in molecular biology from the University of California at Berkeley. As a graduate student, I studied how bovine papillomavirus executes its genetic programme when it infects a cell. This involved a lot of cell culture, DNA binding experiments, affinity columns and electron microscopy. As a postdoc at UC San Francisco, I was more of a developmental biologist, studying how fruit fly embryos establish their body segments. I worked on that project for three years before deciding to leave the bench.

Function of Fos proteins in bone cell differentiation

c-Fos, a component of the dimeric transcription factor AP-1, is a key regulator of bone cell growth and differentiation, affecting both osteoblast and osteoclast lineages during normal development and in bone diseases. Overexpression of c-Fos in transgenic mice results in osteoblastic or chondroblastic tumors. Osteosarcoma formation in c-Fos transgenic mice is enhanced in the p53-/-background, where higher numbers of tumors per animal develop with an earlier onset, thus providing a valuable tumor model (Jochum, W. and Wagner, E. F., unpublished observations). Unlike c-Fos, overexpression of Fra-1, a c-Fos-related protein, in transgenic mice did not induce bone tumors. Lack of tumorigenicity in Fra-1 transgenic mice is consistent with the notion that Fra-1 lacks transcriptional activation domains required for cellular transformation. Unexpectedly, however, Fra-1 transgenic mice showed a progressive increase in bone mass. This osteosclerotic phenotype is characterized by an increase in osteoblast surface and osteoid volume. We are currently analyzing the direct involvement of Fra-1 in osteoblast proliferation and differentiation. In contrast, mice lacking c-Fos develop osteopetrosis due to a complete block in osteoclast differentiation. To have an insight into the mechanism by which c-Fos is required for osteoclastogenesis, an in vitro c-fos-/-rescue system was established whereby c-Fos-deficient splenocytes were infected with retroviral vectors expressing various AP-1 components. After culturing the infected cells in osteoclastogenic conditions, rescued osteoclast formation was quantitated by counting cell numbers expressing osteoclast markers such as tartrate-resistant acid phosphatase (TRAP) and calcitonin receptor. In addition, bone resorption was measured on bovine bone slices. Surprisingly, Fra-1, which lacks transactivation domains detectable in c-Fos, rescued osteoclast differentiation even more efficiently than c-Fos in vitro. Fra-2 and FosB rescued to a lesser extent than c-Fos. Unlike Fos proteins, however, none of the Jun proteins (c-Jun, JunB, and JunD) rescued the differentiation block. Furthermore, the above-mentioned fra-1 transgene, when crossed into mice lacking c-Fos, allowed osteoclast differentiation to proceed and rescued the osteopetrotic phenotype. Regarding the specific role of Fos proteins in osteoclast formation, a number of conclusions can be drawn from our present analyses. First, Fra-1 is the most potent osteoclastogenic Fos protein; introduction of Fra-1 into osteoclastogenic cell lines further increased their differentiation potentials. Second, the fra-1 gene carries AP-1 sites and is transcriptionally activated by c-Fos in the macrophage/osteoclast lineage. Although the fra-1 gene can be activated in a c-Fos-independent manner in many other cell types, transcriptional activation of fra-1 in osteoclast precursors seems to require c-Fos. This implies that c-Fos knock-out mice may be equivalent to c-Fos and Fra-1 double knock-out in the macrophage/osteoclast lineage, leading to an osteopetrotic phenotype. Note that Fra-1 knock-out mice are embryonic lethal due to a placental defect. (Schreiber, M. and Wagner, E. F., unpublished observations). Osteoclastogenic activity of c-Fos in wild-type mice may be either partially or fully mediated by Fra-1. Third, c-Fos is known to carry a major transactivation domain in its COOH-terminal part, which responds to Ras signaling and is critical for transformation. A detailed structure-function analysis showed that COOH-terminal parts of Fos proteins are dispensable for osteoclast differentiation. Further analyses are necessary to identify the mechanisms by which Fra-1, or c-Fos lacking transactivation domains, efficiently activate target genes required for osteoclast differentiation in the absence of c-Fos. One mechanism by which Fos proteins regulate osteoclastspecific gene expression is revealed by the analysis of the TRAP promoter region. Transient transfection and DNA binding experiments revealed juxtaposed functional binding sites for transcription factors NF-AT (nuclear factor of activated T cells) and AP-1. Overexpression of full-length NF-AT3 and Fra-1 activated TRAP promoter-reporter constructs in nonhematopoietic cell lines treated with calcium ionophore and phorbol 12-myristate 13-acetate (PMA). Moreover, ectopic expression of NF-AT3 in peritoneal macrophages and in a macrophage cell line induced expression of the endogenous TRAP gene, suggesting that NF-AT is indeed a key player in osteoclast-specific TRAP gene regulation. Identification of additional AP-1 target genes during osteoclastogenesis as well as further experiments aiming to define a causal role of the transcription factor NF-AT in osteoclast-specific gene expression are important avenues for future research.

Identification and Characterization of a Haploid Germ Cell-specific Nuclear ProteinKinase (Haspin) in Spermatid Nuclei and Its Effects on Somatic Cells

We have cloned the entire coding region of a mouse germ cell-specific cDNA encoding a unique protein kinase whose catalytic domain contains only three consensus subdomains (I-III) instead of the normal 12. The protein possesses intrinsic Ser/Thr kinase activity and is exclusively expressed in haploid germ cells, localizing only in their nuclei, and was thus named Haspin (for haploid germ cell-specific nuclear protein kinase). Western blot analysis showed that specific antibodies recognized a protein of Mr 83,000 in the testis. Ectopically expressed Haspin was detected exclusively in the nuclei of cultured somatic cells. Even in the absence of kinase activity, however, Haspin caused cell cycle arrest at G1, resulting in growth arrest of the transfected somatic cells. In a DNA binding experiment, approximately one-half of wild-type Haspin was able to bind to a DNA-cellulose column, whereas the other half was not. In contrast, all of the deletion mutant Haspin that lacked autophosphorylation bound to the DNA column. Thus, the DNA-binding activity of Haspin may, in some way, be associated with its kinase activity. These observations suggest that Haspin has some critical roles in cell cycle cessation and differentiation of haploid germ cells.

Replication Protein A (RPA) Binding to Duplex Cisplatin-damaged DNA Is Mediated through the Generation of Single-stranded DNA

Replication protein A (RPA) is a heterotrimeric protein composed of 70-, 34-, and 14-kDa subunits that has been shown to be required for DNA replication, repair, and homologous recombination. We have previously shown preferential binding of recombinant human RPA (rhRPA) to duplex cisplatin-damaged DNA compared with the control undamaged DNA (Patrick, S. M., and Turchi, J. J. (1998) Biochemistry 37, 8808-8815). Here we assess the binding of rhRPA to DNA containing site-specific cisplatin-DNA adducts. rhRPA is shown to bind 1.5-2-fold better to a duplex 30-base pair substrate containing a single 1,3d(GpXpG) compared with a 1,2d(GpG) cisplatin-DNA intrastrand adduct, consistent with the difference in thermal stability of DNA containing each adduct. Consistent with these data, a 21-base pair DNA substrate containing a centrally located single interstrand cisplatin cross-link resulted in less binding than to the undamaged control DNA. A series of experiments measuring rhRPA binding and concurrent DNA denaturation revealed that rhRPA binds duplex cisplatin-damaged DNA via the generation of single-stranded DNA. Single-strand DNA binding experiments show that rhRPA binds 3-4-fold better to an undamaged 24-base DNA compared with the same substrate containing a single 1,2d(GpG) cisplatin-DNA adduct. These data are consistent with a low affinity interaction of rhRPA with duplex-damaged DNA followed by the generation of single-stranded DNA and then high affinity binding to the undamaged DNA strand.

The HMG-I/Y protein PF1 stimulates binding of the transcriptional activator GT-2 to the PHYA gene promoter.

The DNA-binding proteins PF1 and GT-2 are factors that bind to different functionally defined, positively acting cis-elements in the PHYA genes of oat and rice, respectively. PF1 is an HMG-I/Y protein, with its cognate cis-element being an AT-rich sequence, designated PE1, whereas GT-2 is a transcriptional activator with twin DNA binding domains that recognize a triplet of GT-boxes in a complex motif designated GTE. To further define the DNA-binding activity of PF1 and to explore potential inter-relationships between the two factors, we have performed a series of in vitro DNA-binding experiments with both PE1 and GTE target sites. The data show that, consistent with its membership of the HMG-I/Y protein family, PF1 can bend DNA when bound to PE1. In addition, PF1 can bind promiscuously, with varying affinity, to other AT-containing motifs, including GTE. When co-incubated with GT-2, PF1 enhances the specific DNA-binding activity of GT-2 toward GTE, the first report of such activity for a plant HMG-I/Y protein. This enhancement takes place without demonstrable physical contact between the two proteins, suggesting the possibility of a novel, indirect mechanism of recruitment involving DNA target-site pre-conditioning. The evidence indicates therefore that PF1 and GT-2 do not perform functionally equivalent roles in positively regulating oat and rice PHYA gene expression. However, the data suggest the possibility that PF1 may act as an architectural factor, promiscuously recognizing a spectrum of AT-containing elements in plant promoters, with the general function of catalyzing enhanced binding of conventional cognate transcriptional regulators to these elements via DNA bending.

A trans-acting peptide activates the yeast a1 repressor by raising its DNA-binding affinity.

The cooperative binding of gene regulatory proteins to DNA is a common feature of transcriptional control in both prokaryotes and eukaryotes. It is generally viewed as a simple energy coupling, through protein-protein interactions, of two or more DNA-binding proteins. In this paper, we show that the simple view does not account for the cooperative DNA binding of a1 and alpha2, two homeodomain proteins from budding yeast. Rather, we show through the use of chimeric proteins and synthetic peptides that, upon heterodimerization, alpha2 instructs a1 to bind DNA. This change is induced by contact with a peptide contributed by alpha2, and this contact converts a1 from a weak to a strong DNA-binding protein. This explains, in part, how high DNA-binding specificity is achieved only when the two gene regulatory proteins conjoin. We also provide evidence that features of the a1-alpha2 interaction can serve as a model for other examples of protein-protein interactions, including that between the herpes virus transcriptional activator VP16 and the mammalian homeodomain-containing protein Oct-l.

Kinetic and Thermodynamic Analysis of Mutant Type II DNA Topoisomerases That Cannot Covalently Cleave DNA

DNA topoisomerase II catalyzes two different chemical reactions as part of its DNA transport cycle: ATP hydrolysis and DNA breakage/religation. The coordination between these reactions was studied using mutants of yeast topoisomerase II that are unable to covalently cleave DNA. In the absence of DNA, the ATPase activities of these mutant enzymes are identical to the wild type activity. DNA binding stimulates the ATPase activity of the mutant enzymes, but with steady-state parameters different from those of the wild type enzyme. These differences were examined through DNA binding experiments and pre-steady-state ATPase assays. One mutant protein, Y782F, binds DNA with the same affinity as wild type protein. This mutant topologically traps one DNA circle in the presence of a nonhydrolyzable ATP analog under the same conditions that the wild type protein catenates two circles. Rapid chemical quench and pulse-chase ATPase experiments reveal that the mutant proteins bound to DNA have the same sequential hydrolysis reaction cycle as the wild type enzyme. Binding of ATP to the mutants is not notably impaired, but hydrolysis of the first ATP is slower than for the wild type enzyme. Models to explain these results in the context of the entire DNA topoisomerase II reaction cycle are discussed.

Bipartite determinants of DNA-binding specificity of plant basic leucine zipper proteins.

The basic leucine zipper (bZIP) proteins are one of the largest and most conserved groups of eukaryotic transcription factors/repressors. Two major subgroups among the plant bZIP proteins have been identified as G-box (CCACGTGG) or C-box (TGACGTCA) binding proteins based on their DNA binding specificity and the amino acid sequences of their basic regions. We have investigated how plant bZIP proteins determine their DNA binding specificity by mutation of the basic domain of the G-box-binding protein EmBP-1. Four subregions of the EmBP-1 basic domain that differ from the C-box-binding protein TGA1a were substituted singly or in combination with the corresponding regions of TGA1a. DNA binding experiments with the mutant proteins demonstrated that binding specificity of plant bZIP proteins is determined independently by two regions, the core basic region and the hinge region. These two regions have an additive effect on DNA binding specificity. PCR-assisted binding-site selections using key mutants demonstrated that only G-box and C-box binding specificity can be generated by combinations of amino acids in the basic domains of EmBP-1 and TGA1a. These results suggest that factorial contributions of the amino acid residues in the basic domain combine to determine DNA-binding specificity of bZIP proteins.

Stimulation of hyaluronan synthesis by tumor necrosis factor-α is mediated by the p50/p65 NF–κB complex in MRC-5 myofibroblasts

The lesions of fibrocontractive diseases result from an excessive myofibroproliferative response to numerous forms of inflammatory stimuli, which elicit the net deposition of extracellular matrix (ECM) in the interstitium of the affected tissue. Hyaluronan (HA), reported to be a key player supporting cellular migration and adherence, is a major component of ECM that undergoes dynamic regulation during inflammation. The molecular regulation of HA biosynthesis by inflammatory cytokines on myofibroblasts is not yet completely understood. Here we report the biochemical characteristics of the lung myofibroblast cell line MRC-5, and we demonstrate that the production of HA by this cell line is inducible by the proinflammatory cytokine, tumor necrosis factor-α (TNF-α), at the message level of HA synthase (HAS). In TNF-α-stimulated MRC-5 cells, DNA-binding and competition experiments indicated that the predominant NF–κB binding activity detected with nuclear extract-stimulated cells is mediated by the p50/p65 complex. Using antisense oligonucleotides, we confirmed that the TNF-α-stimulation of HA synthesis by MRC-5 cells is dependent on the activation of the p50/p65 NF–κB complex. These findings indicate that TNF-α production within inflamed tissues may enhance the HA synthesis via the transcriptional induction of HAS on myofibroblasts, thereby providing a provisional matrix for supporting cellular migration and adhesion, and that the p50/p65 NF–κB complex that plays an important role in the regulation of HA production by TNF-α might be an appropriate target for therapeutic compounds to treat tissue fibrosis accompanied by inflammation.

The ClpB ATPase of Streptomyces albus G belongs to the HspR heat shock regulon.

The clpB gene of Streptomyces albus was cloned by polymerase chain reaction (PCR) using degenerate oligonucleotides. Transcriptional analysis showed that the clpB gene was heat induced. Primer extension identified a transcription start site preceded by typical vegetative -10 and -35 hexamer sequences. The Streptomyces HspR repressor is known to bind to three inverted repeat motifs (IR1, IR2, IR3) upstream from the S. coelicolor dnaK operon. We identified an inverted repeat motif identical to IR3 upstream from the S. albus clpB gene. DNA-binding experiments showed that HspR regulates clpB transcription by interacting directly with this motif. Streptomyces albus is the first Gram-positive organism for which the co-regulation of DnaK and ClpB has been described. Such co-regulation suggests that there is a physiological relationship between these two proteins in this bacterium. Genes similar to hspR were also identified in Mycobacterium leprae, M. tuberculosis and in bacteria unrelated to the actinomycetales order, such as Helicobacter pylori and Aquifex aeolicus. HspR binding sites were found in these bacteria upstream from various heat shock genes, suggesting that these genes are regulated by HspR. The HspR binding site, here called HAIR (HspR associated inverted repeat), has the consensus sequence CTTGAGT N7 ACTCAAG.

Zinc-finger proteins: the classical zinc finger emerges in contemporary plant science.

TFIIIA-type zinc fingers have been found in a number of eucaryotic transcription factors as DNA-binding motifs. In plants, as many as 30 proteins have been reported that have either one, two, three or four zinc fingers. Plant zinc-finger proteins are characterized by long spacers of diverse lengths between adjacent fingers and a highly conserved sequence, QALGGH, located within a putative DNA-contacting surface of each finger. In vitro DNA-binding experiments with two-fingered proteins of petunia have revealed that these proteins bind to target DNA sequences in a manner that is distinctive from that of their animal counterparts: (1) they specifically recognize the spacing between two core sites in target DNA, (2) they have a unique base-determinant position. Regulatory functions have been assigned to some of the TFIIIA-type zinc finger proteins in Arabidopsis, petunia and chinese cabbage. SUPERMAN, AtZFP1, PetSPL3 and BcZFP1 have been implicated in the developmental regulation of various floral and vegetative organs, presumably through the control of cell division and/or expansion in particular cell types. Several anther-specific zinc-finger proteins in petunia are presumed to be involved in the regulation of gametogenesis in both reproductive and non-reproductive tissues of anther. STZ and ZPT2-2 are implicated in the response of plants to or tolerance for various stresses.

Residues in the Swi5 zinc finger protein that mediate cooperative DNA binding with the Pho2 homeodomain protein.

The Swi5 zinc finger and the Pho2 homeodomain DNA-binding proteins bind cooperatively to the HO promoter. Pho2 (also known as Bas2 or Grf10) activates transcription of diverse genes, acting with multiple distinct DNA-binding proteins. We have performed a genetic screen to identify amino acid residues in Swi5 that are required for synergistic transcriptional activation of a reporter construct in vivo. Nine unique amino acid substitutions within a 24-amino-acid region of Swi5, upstream of the DNA-binding domain, reduce expression of promoters that require both Swi5 and Pho2 for activation. In vitro DNA binding experiments show that the mutant Swi5 proteins bind DNA normally, but some mutant Swi5 proteins (resulting from SWI5* mutations) show reduced cooperative DNA binding with Pho2. In vivo experiments show that these SWI5* mutations sharply reduce expression of promoters that require both SWI5 and PHO2, while expression of promoters that require SWI5 but are PHO2 independent is largely unaffected. This suggests that these SWI5* mutations do not affect the ability of Swi5 to bind DNA or activate transcription but specifically affect the region of Swi5 required for interaction with Pho2. Two-hybrid experiments show that amino acids 471 to 513 of Swi5 are necessary and sufficient for interaction with Pho2 and that the SWI5* point mutations cause a severe reduction in this two-hybrid interaction. Analysis of promoter activation by these mutants suggests that this small region of Swi5 has at least two distinct functions, conferring specificity for activation of the HO promoter and for interaction with Pho2.