A highly sensitive plant hybrid protein assay system based on the Spm promoter and TnpA protein for detection and analysis of transcription activation domains.
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.
Schläppi
M
M
Carnegie Institution of Washington, Department of Embryology, Baltimore, MD 21210, USA.
Raina
R
R
Fedoroff
N
N
D004251:DNA Transposable Elements / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000235:genetics*; D005656:Fungal Proteins / Q000235:genetics; D017405:Herpes Simplex Virus Protein Vmw65 / Q000235:genetics; D010940:Plant Proteins / Q000235:genetics*; D011401:Promoter Regions, Genetic / Q000235:genetics; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins; D015534:Trans-Activators / Q000032:analysis; D014157:Transcription Factors; D015533:Transcriptional Activation; D003313:Zea mays / Q000235:genetics
D004251:DNA Transposable Elements; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; C052130:GAL4 protein, S cerevisiae; D017405:Herpes Simplex Virus Protein Vmw65; D010940:Plant Proteins; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; D015534:Trans-Activators; D014157:Transcription Factors