Abstract
TnpA is a multifunctional DNA binding protein encoded by the maize Suppressor-mutator (Spm) transposable element. TnpA is required for transposition and is a repressor of the unmethylated Spm promoter. While analyzing protein domains using a yeast GAL4-based hybrid system in transiently transformed tobacco cells, we found that TnpA represses the >10-fold transcriptional activation observed when the GAL4 DNA-binding domain is used alone. By contrast, compared to the backgroundless TnpA DNA-binding domain alone, 33-to 45-fold activation of the Spm promoter was observed when the VP16 activation domain was fused to it. TnpA-binding sites, but no TATA box, were required for transcription activation. Among the TnpA deletion derivatives tested, those retaining the coding sequences for the DNA-binding and protein dimerization domains gave the highest level of transcription activation when fused with the VP16 activation domain. The TnpA gene and TnpA-binding sites in the short Spm promoter therefore provide a novel, highly sensitive single-hybrid system for identifying and studying plant transcription activation domains in plant cells.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
An G, Mitra A, Choi HK, Costa MA, An K, Thornburg RW, Ryan CA: Functional analysis of the 3′ control region of the potato wound-inducible proteinase II gene. Plant Cell 1: 115–122 (1989).
Cook D, Fedoroff N: Regulation of Spm promoter activity by Spm-encoded tnpA gene product and DNA methylation. Maize Genet Coop Newsl 66: 11–12 (1992).
deWet JR, Wood KV, DeLuca M, Helinski DR, Subramani S: Firefly luciferase gene: structure and expression in mammalian cells. Mol Cell Biol 7: 25–37 (1987).
Fedoroff N, Schläppi M, Raina R: Epigenetic regulation of the maize Spm transposon. BioEssays 17: 291–297 (1995).
Fisher JA, Giniger E, Maniatis T, Ptashne M: GAL4 activates transcription in Drosophila. Nature 332: 853–856 (1988).
Frey M, Reinecke J, Grant S, Saedler H, Gierl A: Excision of the En/Spm transposable element of Zea mays requires two element-encoded proteins. EMBO J 9: 4037–4044 (1990).
Gardner RC, Howarth AJ, Hahn P, Brow-Luedi M, Wilson TMA: The complete nucleotide sequence of an infectious clone of cauliflower mosaic virus by M13mp7 shot-gun sequencing. Nucl Acids Res 15: 3257–3273 (1981).
Gierl A, Lütticke S, Saedler H: TnpA product encoded by the transposable element En-1 of Zea mays is a DNA binding protein. EMBO J 7: 4045–4053 (1988).
Giniger E, Varnum SM, Ptashne M: Specific DNA binding of Gal4, a positive regulatory protein of yeast. Cell 40: 767–774 (1985).
Goff SA, Cone KC, Fromm ME: Identification of functional domains in the maize transcriptional activator C1: comparison of wild-type and dominant inhibitor proteins. Genes Devel 5: 298–309 (1991).
Goff SA, Cone KC, Chandler VL: Functional analysis of the transcriptional activator encoded by the maize B gene: evidence for a direct functional interaction between two classes of regulatory proteins. Genes Devel 6: 864–875 (1992).
Ma J, Przibilla E, Hu J, Bogorad L, Ptashne M: Yeast activators stimulate plant gene expression. Nature 334: 631–633 (1988).
Ma J, Ptashne M: A new class of yeast transcriptional activators. Cell 51: 113–119 (1987).
Masson P, Surosky R, Kingsbury J, Fedoroff NV: Genetic and molecular analysis of the Spm-dependent a-m2 alleles of the maize a locus. Genetics 177: 117–137 (1987).
Masson P, Fedoroff NV: Mobility of the maize Suppressormutator element in transgenic tobacco cells. Proc Natl Acad Sci USA 86: 2219–2223 (1989).
Masson P, Strem M, Fedoroff N: The tnpA and tnpD gene products of the Spm element are required for transposition in tobacco. Plant Cell 3: 73–85 (1991).
Raina R, Cook D, Fedoroff N: Maize Spm transposable element has an enhancer-insensitive promoter. Proc Natl Acad Sci USA 90: 6355–6359 (1993).
Roth BA, Goff SA, Klein TM, Fromm ME: C1-and R-dependent expression of the maize Bzl gene requires sequences with homology to mammalian myb and myc binding sites. Plant Cell 3: 317–325 (1991).
Russell JA, Roy MK, Sandford JC: Major improvements in biolistic transformation of suspension-cultured tobacco cells. In Vitro Cell Devel Biol 28: 97–105 (1992).
Schläppi M, Raina R, Fedoroff N: Epigenetic regulation of the maize Spm transposable element: novel activation of a methylated promoter by TnpA. Cell 77: 427–437 (1994).
Trentmann SM, Saedler H, Gierl A: The transposable element En/Spm-encoded TNPA protein contains a DNA binding and a dimerization domain. Mol Gen Genet 238: 201–208 (1993).
Triezenberg SJ, Kingsbury RC, McKnight SL: Functional dissection of VP16, the transactivator of herpes simplex virus intermediate early gene expression. Genes Devel 2: 718–729 (1988).
Unger E, Pearson RL, Schmidt RJ, Bowen B, Roth BA: Dominant negative mutants of opaque2 suppress transactivation of a 22-kD zein promoter by opaque2 in maize endosperm cells. Plant Cell 5: 831–841 (1993).
Webster N, Jin JR, Gree S, Hollis M, Chambon P: The yeast UAS G is a transcriptional enhancer in human HeLa cells in the presence of the GAL4 trans-activator. Cell 52: 169–178 (1988).
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Schläppi, M., Raina, R. & Fedoroff, N. A highly sensitive plant hybrid protein assay system based on the Spm promoter and TnpA protein for detection and analysis of transcription activation domains. Plant Mol Biol 32, 717–725 (1996). https://doi.org/10.1007/BF00020212
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF00020212