Faithful transcription initiation of fibroin gene in a homologous cell-free system reveals an enhancing effect of 5′ flanking sequence far upstream

Abstract

For the faithful initiation of transcription of exogenously added fibroin gene, a crude cellular extract was prepared from the posterior silk glands where fibroin gene is specifically transcribed. The faithful initiation was demonstrated by the assay with use of truncated fibroin gene templates and confirmed by nuclease S1 mapping. The transcription initiation of fibroin gene was detected also in the extract from the middle silk glands where fibroin gene is not transcribed, indicating that an expected suppression machinery for the gene was left behind during the extraction procedures. The promoter sequence of fibroin gene required in these homologous extracts (the nucleotide position −29 to +6) is the same as that in a heterologous HeLa cell extract. This result guarantees a limited usefulness of the HeLa cell extract in determining promoter sequence or analyzing general transcription machinery. In the homologous extracts, but not in the heterologous extract, however, it has been shown for the first time in vitro that the 5′ flanking sequence upstream from −74 enhances the transcription initiation of fibroin gene. In addition, a remarkable fibroin gene preference in transcription over mouse β globin and adenovirus 2 major late genes was observed in the homologous extracts. The fibroin gene preference is partly caused by the sequence element upstream from −74 of fibroin DNA.