Efficient and strand-selective in vitro transcription initiation by purified RNA polymerase II from a unique site of the fibroin gene.

Using purified RNA polymerase II and the cloned fibroin gene of supercoiled form, we found a novel in vitro transcription initiation site. An analysis by S1 nuclease mapping showed that the transcripts synthesized by RNA polymerase II purified from three different sources gave primarily a common protection band, the 5' terminus of which was located at a specific site about the nucleotide position +25 of the fibroin gene. Such a transcript was not detected in the in vivo RNA; however, this site-specific transcription revealed a coding-strand selectivity and a high efficiency accounting for nearly 90% of total in vitro RNA synthesis. An analysis using a series of 5' deletion mutants of the fibroin gene indicates that this transcription initiation depends on a unique DNA sequence about the nucleotide position +25, which includes an inverted repeat and a sequence homologous to that around the in vivo initiation site of the fibroin gene.