Point mutation in the TATA box curtails expression of sea urchin H2A histone gene in vivo

Abstract

In most, but not all, eukaryotic genes transcribed by polymerase B (II)1–5, the site of initiation of transcription is preceded by a TATAATAAT sequence4, commonly referred to as the Goldberg–Hogness6 or TATA box, which seems to be required for the generation of faithful 5′ termini of mRNAs7–14. For all genes except one9,12,15–17, deletion of the TATA box reduces the rate of mRNA synthesis7,10,11,13,18–23. Point mutations in the TATA box of the chick conalbumin gene diminish transcription in vitro 20–40-fold21,24. The question remained of whether this effect would persist in vivo where far upstream sequences dramatically modulate gene expression7–11,25,26. Unfortunately the chicken conalbumin gene is poorly expressed when introduced into eukaryotic cells in culture (B.W., unpublished results) or into Xenopus oocytes (S. Swany, personal communication). We have therefore constructed a new gene unit with a chimaeric promoter region by introducing the conalbumin wild-type or mutated TATA box region into the sea urchin H2A histone gene, which is known to be efficiently expressed in Xenopus oocytes27–29. We report here that with both the wild-type and mutated chimaeric promoters, initiation of transcription occurs at a novel initiation site about 26 nucleotides downstream from the TATA or TAGA boxes, but in the mutant case, the amount of specific transcripts is decreased fivefold.