IN-VITRO TRANSCRIPTION OF A DROSOPHILA U1 SMALL NUCLEAR-RNA GENE REQUIRES TATA BOX-BINDING PROTEIN AND 2 PROXIMAL CIS-ACTING ELEMENTS WITH STRINGENT SPACING REQUIREMENTS

Transcription of a Drosophila U1 small nuclear RNA gene was functional ly analyzed in cell extracts derived from 0- to 12-h embryos. Two prom oter elements essential for efficient initiation of transcription in v itro by RNA polymerase 11 were identified. The first, termed PSEA, is located between positions -41 and -61 relative to the transcription st art site, is crucial for promoter activity, and is the dominant elemen t for specifying the transcription initiation site. PSEA thus appears to be functionally homologous to the proximal sequence element of vert ebrate small nuclear RNA genes. The second element, termed PSEB, is lo cated at positions -25 to -32 and is required for an efficient level o f transcription initiation because mutation of PSEB, or alteration of the spacing between PSEA and PSEB, severely reduced transcriptional ac tivity relative to that of the wild-type promoter. Although the PSEB s equence does not have any obvious sequence similarity to a TATA box, c onversion of PSEB to the canonical TATA sequence dramatically increase d the efficiency of the U1 promoter and simultaneously relieved the re quirement for the upstream PSEA. Despite these effects, introduction o f the TATA sequence into the U1 promoter had no effect on the choice o f start site or on the RNA polymerase II specificity of the promoter. Finally, evidence is presented that the TATA box-binding protein is re quired for transcription from the wild-type U1 promoter as well as fro m the TATA-containing U1 promoter.