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 TATA^A_TA^A_T sequence⁴, commonly referred to as the Goldberg–Hogness⁶ or TATA box, which seems to be required for the generation of faithful 5′ termini of mRNAs^7–14. For all genes except one^9,12,15–17, deletion of the TATA box reduces the rate of mRNA synthesis^{7,10,11,13,18–23}. Point mutations in the TATA box of the chick conalbumin gene diminish transcription in vitro 20–40-fold^21,24. The question remained of whether this effect would persist in vivo where far upstream sequences dramatically modulate gene expression^7–11,25,26. Unfortunately the chicken conalbumin gene is poorly expressed when introduced into eukaryotic cells in culture (B.W., unpublished results) or into Xenopus oocytes (S. Swany, personal communication).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 oocytes^27–29. 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.