Reconstitution of transcription from the human U6 small nuclear RNA promoter with eight recombinant polypeptides and a partially purified RNA polymerase III complex

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