TRANSCRIPTION OF THE SEA-URCHIN U6 GENE IN-VITRO REQUIRES A TATA-LIKE BOX, A PROXIMAL SEQUENCE ELEMENT, AND SEA-URCHIN USF, WHICH BINDS AN ESSENTIAL E-BOX

The tandemly repeated gene set encoding the sea urchin U6 gene has been cloned from the sea urchin Strongylocentrotus purpuratus. The U6 gene is transcribed by RNA polymerase III in a sea urchin nuclear extract. Like that of the vertebrate U6 genes, transcription of the sea urchin U6 gene does not require any internal sequences or 3' sequences but requires only 5' flaking sequences. Only 88 nucleotides of 5' flanking sequence are required for maximal expression in vitro. Mutagenesis experiments demonstrated the requirement for three elements, a CACGTG element at -80, a proximal sequence element at about -55, and the TATA-like box at -25. The major protein in sea urchin extracts that interacts with the CACGTG element is sea urchin USF, and immunodepletion of sea urchin USF greatly reduces transcription. The USF binding site in the U6 gene is highly homologous (11 of 13 nucleotides) with the USF binding sites found in the promoter of the S. purpuratus spec genes.