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 tile 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.