Initiator-dependent transcription in vitro by a wheat germ chromatin extract

The development of plant in vitro transcription systems transcribing faithfully and efficiently from a broad range of plant nuclear promoters has remained a challenge. We examined the nucleotide sequence requirements for faithful and efficient transcription in a wheat germ chromatin extract (Yamazaki et al., Plant Mol Biol Rep 8: 114-123). The wheat germ chromatin extract was tested with a series of chimeric promoter constructs containing plant promoter sequences upstream from the TATA box, TATA boxes, and cap-site sequences (from -10 to +14, relative to the major in vivo initiation site) in different combinations. The plant extract transcribed faithfully from several chimeric promoters containing the capsite sequence of the parsley chalcone synthase promoter. The transcription was sensitive to the RNA polymerase II-specific inhibitor alpha-amanitin and was only dependent on the chalcone synthase cap-site sequence which therefore fulfils the operational criteria for a plant initiator element. Mutations of the putative chalcone synthase initiator element defined a core sequence '5'TAACAAC' around the initiation site that was necessary for efficient transcription in vitro. In contrast to the extract, purified wheat germ RNA polymerase II showed no preference for transcription from the major chalcone synthase in vivo initiation site.