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A single nucleotide exchange in a promoter region located immediately upstream of the CAAT box of the spinach photosynthesis gene AtpC (gene product is subunit γ of the chloroplast ATP synthase) prevents the formation of a secondary structure and causes an unregulated, constitutive high level of expression (Kusnetsov, V., Landsberger, M., Oelmüller, R. (1999)J. Biol. Chem. 274, 36009–36014). We have isolated cDNAs for ATPC-2, a new polypeptide with homologies to pro- and eukaryotic helicases, which specifically binds to this promoter region. Binding of ATPC-2 competes strongly with that of a CAAT box binding factor (CBF), consistent with the idea that both complexes cannot be formed simultaneously because of sterical reasons. In gel mobility shift assays, high binding activities of ATPC-2 and low binding activities of CBF were observed with nuclear extracts from tissue with low AtpC expression levels, and the opposite was observed with extracts from tissues with high AtpCexpression levels. Binding of ATPC-2 to the mutant sequence, which directs a constitutively high level expression in vivo and prevents the formation of a secondary structure in vitro,is significantly weaker than binding to the wild-type sequence. Again, the opposite results were obtained for the CBF. Thus, we conclude that the assembly of the CBF·DNA complex stimulates transcription of AtpC and that CBF binding is prevented if ATPC-2 is bound to the promoter region. The novel mechanism of gene regulation and the role of the helicase-like protein ATPC-2 as a potential transcriptional repressor is discussed in relation to its modular structure. A single nucleotide exchange in a promoter region located immediately upstream of the CAAT box of the spinach photosynthesis gene AtpC (gene product is subunit γ of the chloroplast ATP synthase) prevents the formation of a secondary structure and causes an unregulated, constitutive high level of expression (Kusnetsov, V., Landsberger, M., Oelmüller, R. (1999)J. Biol. Chem. 274, 36009–36014). We have isolated cDNAs for ATPC-2, a new polypeptide with homologies to pro- and eukaryotic helicases, which specifically binds to this promoter region. Binding of ATPC-2 competes strongly with that of a CAAT box binding factor (CBF), consistent with the idea that both complexes cannot be formed simultaneously because of sterical reasons. In gel mobility shift assays, high binding activities of ATPC-2 and low binding activities of CBF were observed with nuclear extracts from tissue with low AtpC expression levels, and the opposite was observed with extracts from tissues with high AtpCexpression levels. Binding of ATPC-2 to the mutant sequence, which directs a constitutively high level expression in vivo and prevents the formation of a secondary structure in vitro,is significantly weaker than binding to the wild-type sequence. Again, the opposite results were obtained for the CBF. Thus, we conclude that the assembly of the CBF·DNA complex stimulates transcription of AtpC and that CBF binding is prevented if ATPC-2 is bound to the promoter region. The novel mechanism of gene regulation and the role of the helicase-like protein ATPC-2 as a potential transcriptional repressor is discussed in relation to its modular structure. CAAT box binding factor glutathione S-transferase The plastid ATP synthase of higher plants and algae consists of nine different subunits, and three of them are encoded by the nuclear genes AtpC, AtpD, and AtpG (the gene products are the subunits γ and δ of the CF1 moiety and CFoII) (2Herrmann R.G. Oelmüller R. Bichler J. Schneiderbauer A. Steppuhn J. Wedel N. Tyagi A.K. Westhoff P. Herrmann R.G. Larkins B.A. Plant Molecular Biology. 2. Plenum Press, New York1991: 411-427Google Scholar). Comparable with other nuclear-encoded genes for plastid proteins, expression of the spinach ATP synthase genes and chimeric promoter::reporter gene fusions in transgenic tobacco is strongly regulated by light, phytohormones, in particular cytokinins, or the stage of the plastids in cells in which these genes are expressed (cf. Refs. 3Bolle C. Sopory S. Lübberstedt T. Klösgen R.B. Herrmann R.G. Oelmüller R. Plant Physiol. 1994; 105: 1355-1364Crossref PubMed Scopus (38) Google Scholar, 4Bolle C. Kusnetsov V.V. Herrmann R.G. Oelmüller R. Plant J. 1996; 9: 21-30Crossref PubMed Scopus (34) Google Scholar, 5Kusnetsov V.V. Oelmüller R. Sarwat M.I. Porfirova S.A. Cherepneva G.N. Herrmann R.G. Kulaeva O.N. Planta. 1994; 194: 318-327Crossref Scopus (98) Google Scholar, 6Kusnetsov V.V. Bolle C. Lübberstedt T. Sopory S. Herrmann R.G. Oelmüller R. Mol. Gen. Genet. 1996; 252: 631-639PubMed Google Scholar, 7Oelmüller R. Schneiderbauer A. Herrmann R.G. Kloppstech K. Mol. Gen. Genet. 1995; 246: 478-484Crossref PubMed Scopus (19) Google Scholar). However, unusually for photosynthesis genes, the essential cis-elements determining the regulated expression of AtpC are positioned in close vicinity to the respective transcription start sites (4Bolle C. Kusnetsov V.V. Herrmann R.G. Oelmüller R. Plant J. 1996; 9: 21-30Crossref PubMed Scopus (34) Google Scholar), and crucial nucleotides for the regulated expression appear to be located immediately upstream of the CAAT box (4Bolle C. Kusnetsov V.V. Herrmann R.G. Oelmüller R. Plant J. 1996; 9: 21-30Crossref PubMed Scopus (34) Google Scholar). This region also forms a complex with a CAAT box binding factor, CBF1 (1Kusnetsov V. Landsberger M. Oelmüller R. J. Biol. Chem. 1999; 274: 36009-36014Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). However, in contrast to many CBFs from metazoa (8Maity S.N. de Crombrugghe B. Trends Biochem. Sci. 1998; 23: 174-178Abstract Full Text Full Text PDF PubMed Scopus (312) Google Scholar, 9McNabb D.S. Xing Y. Guarente L. Genes Dev. 1994; 9: 47-58Crossref Scopus (234) Google Scholar, 10Sinha S. Maity S.N. Lu J. de Crombrugghe B. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 1624-1628Crossref PubMed Scopus (251) Google Scholar) the binding activity inin vitro assays is regulated, and the binding activities correspond to the promoter activities in vivo (1Kusnetsov V. Landsberger M. Oelmüller R. J. Biol. Chem. 1999; 274: 36009-36014Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). Surprisingly, a single nucleotide exchange uncoupled the promoter activity from regulatory pathways and resulted in a high constitutive expression. This indicates that low promoter activities (e.g. in darkness or in photobleached tissue) are caused by an inhibitory effect that is no longer active under conditions of high promoter activities, e.g. in light or after cytokinin treatments. Here we describe the isolation of a DNA that codes for a protein with sequence-specific binding characteristics to this promoter region. We demonstrate that the binding activity is high in tissue with low AtpC expression levels and, vice versa, that this behavior is opposite to the binding features described previously for CBF (1Kusnetsov V. Landsberger M. Oelmüller R. J. Biol. Chem. 1999; 274: 36009-36014Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar), and that the recombinant protein competes with CBF for binding. The data are consistent with a model in which this protein functions as a repressor by preventing the assembly of CBF at the CAAT box and thus repressing AtpC transcription. Tobacco seeds were surface-sterilized, planted on 1/2 Murashige and Skoog medium supplemented with 2% sucrose in the presence or absence of Norflurazon (10−6m) and kept in a cold room in darkness for 2 days to synchronize germination. Seedlings were either kept in darkness for 10 days at 22 °C or they were transferred to white light for 40 h prior to harvesting in the absence or presence of Norflurazon. Alternatively, etiolated seedlings were transferred to cytokinin-containing plates (N 6-benzylaminopurine, 10−5m) in darkness 40 h prior to harvest. Oligonucleotides for wild-type and mutant sequences were annealed (11Lübberstedt T. Oelmüller R. Wanner G. Herrmann R.G. Mol. Gen. Genet. 1994; 242: 602-613Crossref PubMed Scopus (26) Google Scholar) and cloned into the SmaI site of pBSC+ (Stratagene, San Diego) prior to sequencing. Plasmids were chosen in which the 5′-end of the promoter fragment was orientated toward the BamHI site of pBSC+. Thus all promoter fragments had the following 5′- and 3′ extensions: 5′-GATCCCCC[Insert]GGGCTGCA-3′ and 3′-GGGG[Insert]CCCG-5′. After digestion of the plasmids with PstI and BamHI, the BamHI site was filled-in with all four radiolabeled nucleotides and the Klenow enzyme, and the fragments were gel-purified on agarose gels. For the binding studies in the presence of CBF-C (1Kusnetsov V. Landsberger M. Oelmüller R. J. Biol. Chem. 1999; 274: 36009-36014Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar), nuclear protein extracts were isolated from six-week-old tobacco orArabidopsis plants grown in the greenhouse. For physiological studies, seedlings were grown as described above, and the nuclear protein fractions were prepared as described (12Oelmüller R. Bolle C. Tyagi A.K. Niekrawietz N. Breit S. Herrmann R.G. Mol. Gen. Genet. 1993; 237: 261-272Crossref PubMed Scopus (42) Google Scholar). Approximately 190,000 phages of an Arabidopsis thalianacDNA expression library were screened with the radiolabeledAtpC promoter region (−68 to −39; see below). Two positive phage were plaque-purified, and pBSC+ was excised according to manufacturer's instructions (Stratagene) prior to sequencing. “Southwestern” hybridization was performed at 4 °C. The filters were first blocked with blocking buffer by gentle shaking for 3 h (SW buffer, 1% bovine serum albumin, 1.5 mg/ml denatured salmon sperm DNA; SW buffer = 25 mmHEPES-NaOH, pH 7, 6, 50 mm NaCl, 5 mmMgCl2, 1 mm EDTA, 5% v/v glycerol, 1 mm dithiothreitol). After three washing steps (10 min each) with HS buffer (SW buffer with 1 mg/ml denatured salmon sperm DNA), the filters were incubated overnight with SW buffer in the presence of the radioactive DNA fragments. After washing with SW buffer, the signals were visualized on a PhosphorImager (Storm 820, Molecular Dynamics, Krefeld, Germany). The isolated cDNA as well as the cDNA encoding CBF-C (1Kusnetsov V. Landsberger M. Oelmüller R. J. Biol. Chem. 1999; 274: 36009-36014Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar) were N-terminally fused to GST according to the manufacturer's instructions (pGEX vector system from Amersham Pharmacia Biotech). Plasmids encoding GST fusion proteins were transformed into Escherichia coli strain BL21 (DES)pLysS. Cultures were grown overnight at 37 °C in the presence of 40 μg/ml chloramphenicol and 100 μg/ml ampicillin, diluted 100-fold, and grown at 30 °C to an optical density of 0.6 at 600 nm. Protein expression was induced by isopropyl-β-d-thiogalactopyranoside, with a final concentration of 0.5 mm at 27 °C. Cells were harvested by centrifugation (5,000 × g for 10 min at 4 °C) 4 h after induction, resuspended in 1× phosphate-buffered saline, and sonicated. After centrifugation (12,000 × g for 15 min at 4 °C), aliquots of the supernatant were applied directly onto freshly prepared glutathione-Sepharose 4B columns and further processed according to the manufacturer's instructions (Amersham Pharmacia Biotech). The purified GST and GST fusion proteins were separated by SDS-polyacrylamide gel electrophoresis (12.5%) and transferred onto nitrocellulose. Southwestern analysis was performed as described previously (1Kusnetsov V. Landsberger M. Oelmüller R. J. Biol. Chem. 1999; 274: 36009-36014Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar) with the radiolabeled DNA fragments. For gel mobility shift assays, equal amounts of the purified proteins were used. Gel mobility shift assays were performed essentially as described (1Kusnetsov V. Landsberger M. Oelmüller R. J. Biol. Chem. 1999; 274: 36009-36014Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar,12Oelmüller R. Bolle C. Tyagi A.K. Niekrawietz N. Breit S. Herrmann R.G. Mol. Gen. Genet. 1993; 237: 261-272Crossref PubMed Scopus (42) Google Scholar). CBF-C was incubated with nuclear protein extracts (1 μg/μl) in binding buffer (25 mm HEPES/KOH, pH 7, 6, 50 mm KCl, 5 mm MgCl2, 5% glycerol) for 30 min at 37 °C. Finally, the DNA was added and the mixture incubated at 37 °C for 2 h prior to gel electrophoresis. For competition studies, the purified ATPC-2·GST fusion protein, the CBF-C·GST fusion protein, or GST alone was added to the preformed radiolabeled protein·DNA complex and treated as described in the figure legends. In all of the studies the CBF-C fusion was preincubated with nuclear extract as described above, whereas the ATPC-2 fusion was added directly. A double-stranded oligonucleotide from the −69 to −39AtpC region (5′-TTTACCTCCAAAATTCAATGGCCAAAATCT-3′) harboring the expression-relevant AAAAT (bold) and the CAAT (italics) motifs was used initially to screen an expression library fromArabidopsis. Positive signals were obtained only when the screen was performed in the presence of 1 mg/ml nuclear extract. The four isolated cDNAs encoded CBF-C, the subunit C of a new CAAT box binding complex isolated from plants (1Kusnetsov V. Landsberger M. Oelmüller R. J. Biol. Chem. 1999; 274: 36009-36014Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). Therefore, a second screen was initiated with an oligonucleotide in which the CAAT sequence was replaced by GTTA, an oligonucleotide that fails to bind to CBF-C (1Kusnetsov V. Landsberger M. Oelmüller R. J. Biol. Chem. 1999; 274: 36009-36014Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). Two positive phages with significantly lower binding strength were obtained. Both of them also bind to the wild-type sequence harboring the CAAT motif but not to a mutant sequence with a single nucleotide exchange (A → G) in the AAAAT sequence (data not shown; see below). DNA sequence analysis revealed that both phages carried identical DNA insertions that code for the C-terminal part of a novel protein named ATPC-2. After the complete nucleotide sequence of this gene became available in the data banks, we polymerase chain reaction-amplified the complete cDNA from our library and confirmed that the protein is encoded by a genomic sequence without introns (Fig.1). A homologous cDNA was also isolated from our tobacco cDNA library; however, it is not full-length (data not shown). Data bank searches revealed that the deduced amino acid sequence exhibits homologies to DNA helicases from pro- and eukaryotic organisms (Fig. Surprisingly, the homologies were observed to the protein 2 from a DNA from and a protein from and whereas protein with an exhibits sequence the isolated protein also exhibits to proteins acid sequence however, the of these proteins not searches not functions of ATPC-2 that the protein sites for (Fig. that the to the and proteins the well motifs from DNA helicases and The protein as well as with that are in the data box (Fig. the C-terminal of the protein a of amino and amino acid sequence to other (data not shown). This that the to a novel of proteins in and that this of proteins exhibits to the of DNA helicases (Fig. In a indicates that the eukaryotic proteins are to whereas the proteins appear to be (data not shown). the DNA sequence for the formation of the a of mutant sequences for which the transcriptional activity is from chimeric gene fusions in transgenic tobacco (1Kusnetsov V. Landsberger M. Oelmüller R. J. Biol. Chem. 1999; 274: 36009-36014Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar) was for its to bind to ATPC-2 and Both proteins were in coli as GST fusion proteins, isolated first by glutathione and by electrophoresis The isolated proteins were separated on a gel to binding assays (Fig. 3 in the absence or presence of nuclear extracts demonstrate that the wild-type sequence binds to ATPC-2 in both previously described (1Kusnetsov V. Landsberger M. Oelmüller R. J. Biol. Chem. 1999; 274: 36009-36014Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar), binding of CBF-C the of nuclear extracts The mutant sequence with an A → exchange fails to bind to ATPC-2, whereas CBF-C is of binding in the presence of nuclear extracts In the CAAT mutant sequence fails to bind to CBF-C, whereas it bind to ATPC-2 This was confirmed by gel mobility shift that the binding activity is not by the CAAT box 2 and 3 with 4 and and that the binding activity of ATPC-2 to the oligonucleotide with the A → exchange is and A which the sequence but the CAAT box sequence, also fails to bind to ATPC-2 and this indicates that ATPC-2 binding to the AtpC promoter is sequence-specific and nucleotides located 3′ to the we performed competition purified ATPC-2 and CBF with the wild-type oligonucleotide sequence (Fig. A of the ATPC-2·GST fusion protein cannot CBF-C·GST from the whereas the of CBF-C·GST is for (Fig. The is for the opposite if DNA with ATPC-2·GST is used for competition the binding activity cannot be with a of whereas is (Fig. This indicates that both binding activities are and that either ATPC-2 or CBF bind to this promoter region. The assembly of both complexes simultaneously is not for sterical (cf. mobility shift assays with the GST fusion protein 7, and and GST alone 6, and to the wild-type 2 and and mutant AtpC promoter amounts of the following cloned into the SmaI site of and excised with BamHI and PstI were wild-type sequence 4 and without of ATPC-2 and CBF-C for binding to the wild-type sequence. the radiolabeled oligonucleotide with the wild-type sequence was incubated with the CBF-C·GST fusion protein in the presence of nuclear extracts for 30 min at 37 °C. no or a 5 or 10 of protein was and the were incubated at 37 °C for 2 h gel electrophoresis and competition with competition with the that CBF-C·GST was replaced by ATPC-2·GST and vice A of the promoter activity in vivo (1Kusnetsov V. Landsberger M. Oelmüller R. J. Biol. Chem. 1999; 274: 36009-36014Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar, 3Bolle C. Sopory S. Lübberstedt T. Klösgen R.B. Herrmann R.G. Oelmüller R. Plant Physiol. 1994; 105: 1355-1364Crossref PubMed Scopus (38) Google Scholar) and its to bind to ATPC-2 is in extracts from seedlings binding activity to the wild-type whereas extracts from seedlings higher The a low binding activity and a high promoter activity was observed with nuclear extracts from etiolated seedlings treated with cytokinin promoter activity (1Kusnetsov V. Landsberger M. Oelmüller R. J. Biol. Chem. 1999; 274: 36009-36014Abstract Full Text Full Text PDF PubMed Scopus (86) Google and from seedlings treated with the Norflurazon to chloroplast promoter activity (1Kusnetsov V. Landsberger M. Oelmüller R. J. Biol. Chem. 1999; 274: 36009-36014Abstract Full Text Full Text PDF PubMed Scopus (86) Google This is opposite to the that we obtained previously for CBF binding activity with the (1Kusnetsov V. Landsberger M. Oelmüller R. J. Biol. Chem. 1999; 274: 36009-36014Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). low signals were observed when the A → mutant sequence was used for the of the physiological of the this is consistent with the idea that ATPC-2 prevents the assembly of the CBF at promoter and thus functions as a transcriptional of the were performed with ATPC-2 CBF-C from Arabidopsis with nuclear extracts from the AtpC promoter is regulated in a in an Arabidopsis and to that the ATPC-2 repressor exhibits a in both the in the 5 and were with nuclear protein fractions from the results were that the signals were significantly weaker (data not which be caused by a of the nuclear protein with In many eukaryotic motifs are 50 and 100 nucleotides upstream of the transcription start site A. J. A. C. Full Text PDF PubMed Scopus Google Scholar, Full Text PDF PubMed Scopus Google Scholar, A. de Crombrugghe B. J. Biol. Chem. Full Text PDF PubMed Google Scholar, P. J. Mol. Biol. PubMed Scopus Google Scholar). motifs are by The CAAT box sequences in which are to the of transcriptional be the site for and the assembly of CBF at the CAAT motif in to or signals (8Maity S.N. de Crombrugghe B. Trends Biochem. Sci. 1998; 23: 174-178Abstract Full Text Full Text PDF PubMed Scopus (312) Google Scholar, S.N. G. de Crombrugghe B. PubMed Scopus Google Scholar, P. J. U. PubMed Scopus Google Scholar, T. Maity S.N. de Crombrugghe B. 1996; PubMed Google Scholar, J. Biol. Chem. 1993; Full Text PDF PubMed Google Scholar, J. Biol. Chem. 1993; Full Text PDF PubMed Google Scholar, J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar, M. J. 1994; Google Scholar, P. P. J. Biol. Chem. 1993; Full Text PDF PubMed Google Scholar, Y. M. P. J. 1994; PubMed Scopus Google Scholar, J. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar, B. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar, K. S. J. Biol. Chem. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). Here we demonstrate that the binding activity of the CBF to the CAAT motif in the AtpC promoter is by ATPC-2. the promoter is we observed a low binding activity of ATPC-2 and a high binding activity of CBF and vice 5 and ATPC-2 exhibits to the of DNA helicases to Mol. 1999; PubMed Scopus Google Scholar). helicases the binding and of to that in the of the for different forms of helicases a modular structure and sequence of than 100 from and eukaryotic organisms have revealed amino acid the and a as a and motif is for binding. is homologous to the sequence of a DNA and the of the motifs is that the in the structure is in the motifs and and and as well as at the and C Mol. 1999; PubMed Scopus Google Scholar, PubMed Scopus Google Scholar). however, that of the with the to ATPC-2 are significantly than the well helicases and motifs and In these proteins ATPC-2 the of in the C-terminal region following motif a that is not of all DNA helicases but in of as in 2. This that the ATPC-2 to a novel of proteins that are either of helicases or from helicases that have C-terminal with and the proteins are in pro- and eukaryotic no analysis performed on of of and identical modular it is that they all helicases of and are of acid have to in as DNA DNA or transcription. different DNA motifs and DNA as well as and double-stranded DNA Mol. 1999; PubMed Scopus Google Scholar, Y. T. A. T. 1993; PubMed Scopus Google Scholar, Biol. PubMed Scopus Google Scholar, Biol. 1998; PubMed Scopus Google Scholar, Full Text Full Text PDF PubMed Scopus Google Scholar, 1998; 92: Full Text Full Text PDF PubMed Scopus Google Scholar, P. Biol. PubMed Scopus Google Scholar, Y. M. K. A. T. J. Biol. Chem. 1993; 23: Full Text PDF Google Scholar). ATPC-2, a of binding DNA in a sequence-specific and We have previously that the sequence forms a secondary structure in as a DNA whereas the not (1Kusnetsov V. Landsberger M. Oelmüller R. J. Biol. Chem. 1999; 274: 36009-36014Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). Thus, that the sequence-specific a and an site for a polypeptide with the of the and is not it is that a is of Therefore, a model describe the role of ATPC-2 in AtpC gene The AAAAT sequence forms a protein·DNA complex and thus prevents the assembly of in a AtpC transcription. Both cannot bind simultaneously because of binding (1Kusnetsov V. Landsberger M. Oelmüller R. J. Biol. Chem. 1999; 274: 36009-36014Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). This is by data from the which a DNA region of at nucleotides (8Maity S.N. de Crombrugghe B. Trends Biochem. Sci. 1998; 23: 174-178Abstract Full Text Full Text PDF PubMed Scopus (312) Google Scholar). of AtpC as light or repressor binding and thus for the assembly of this is consistent with the that the A → which prevents binding of the repressor but the assembly of CBF (Fig. results in a constitutive promoter vivo (1Kusnetsov V. Landsberger M. Oelmüller R. J. Biol. Chem. 1999; 274: 36009-36014Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). This novel mechanism of gene regulation a new to the that is caused by that the observed in light C. J. Dev. Biol. PubMed Scopus Google Scholar). We and for and for the
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