INITIATOR-DEPENDENT TRANSCRIPTION IN-VITRO BY A WHEAT-GERM CHROMATIN EXTRACT

The development of plant in vitro transcription systems transcribing f aithfully and efficiently from a broad range of plant nuclear promoter s has remained a challenge. We examined the nucleotide sequence requir ements for faithful and efficient transcription in a wheat germ chroma tin extract (Yamazaki et al., Plant Mol Biol Rep 8: 114-123). The whea t 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 pla nt extract transcribed faithfully from several chimeric promoters cont aining the cap-site sequence of the parsley chalcone synthase promoter . The transcription was sensitive to the RNA polymerase II-specific in hibitor alpha-amanitin and was only dependent on the chalcone synthase cap-site sequence which therefore fulfils the operational criteria fo r a plant initiator element. Mutations of the putative chalcone syntha se initiator element defined a core sequence '5'TAACAAC' around the in itiation site that was necessary for efficient transcription in vitro. In contrast to the extract, purified wheat germ RNA polymerase II sho wed no preference for transcription from the major chalcone synthase i n vivo initiation site.