Efficiency of in vitro transcription of Dictyostelium discoideum actin gene is affected by the nucleotide sequence of the transcription initiation region.

Abstract

The actin gene of Dictyostelium discoideum is transcribed faithfully but with very low efficiency in a cell-free system containing HeLa cell extract [Takiya, S., Tabata, T., Iwabuchi, M., Hirose, S., & Suzuki, Y. (1984) J. Biochem. (Tokyo) 95, 1367-1377]. Using the same in vitro system, we determined that the promoter activity of the actin 5 gene is 100-200 times weaker than that of the silkworm fibroin gene. To clarify the cause of the low transcription efficiency, various chimeric genes were constructed from the actin and fibroin genes, and their transcription efficiencies were examined in vitro. Both the TATA box and the transcription initiation site of the two natural genes functioned in the transcription of the chimeric genes, the efficiency of which was especially affected by the transcription initiation region. In chimeric genes having the upstream sequence of the actin gene and a downstream sequence including the transcription initiation site of the fibroin gene, the transcription efficiency was higher than one-third of that of the natural fibroin gene. In chimeric genes having the actin transcription initiation region and an upstream sequence of the fibroin gene, the transcription efficiency was as low as that of the natural actin gene. We concluded that the transcription initiation site is a part of the promoter and an essential region for directing faithful and efficient initiation of gene transcription.