Reconstitution of Transcription from the Human U6 Small Nuclear RNA Promoter with Eight Recombinant Polypeptides and a Partially Purified RNA Polymerase III Complex

Abstract

The human U6 small nuclear (sn) RNA core promoter consists of a proximal sequence element, which recruits the multisubunit factor SNAPc, and a TATA box, which recruits the TATA box-binding protein, TBP. In addition to SNAPc and TBP, transcription from the human U6 promoter requires two well defined factors. The first is hB″, a human homologue of the B″ subunit of yeast TFIIIB generally required for transcription of RNA polymerase III genes, and the second is hBRFU, one of two human homologues of the yeast TFIIIB subunit BRF specifically required for transcription of U6-type RNA polymerase III promoters. Here, we have partially purified and characterized a RNA polymerase III complex that can direct transcription from the human U6 promoter when combined with recombinant SNAPc, recombinant TBP, recombinant hB″, and recombinant hBRFU. These results open the way to reconstitution of U6 transcription from entirely defined components.