RNA-POLYMERASE-III TRANSCRIPTION FROM THE HUMAN U6 AND ADENOVIRUS TYPE-2 VAI PROMOTERS HAS DIFFERENT REQUIREMENTS FOR HUMAN BRF, A SUBUNIT OF HUMAN TFIIIB

Mammalian TFIIIB can be separated into two fractions required for tran scription of the adenovirus type 2 VAI gene, which have been designate d 0.38M-TFIIIB and 0.48M-TFIIIB. While 0.48M-TFIIIB has not been chara cterized, 0.38M-TFIIIB corresponds to a TBP-containing complex. We des cribe here the purification of this complex, which consists of TBP and a closely associated polypeptide of 88 kDa, and the isolation of a cD NA corresponding to the 88-kDa polypeptide. The predicted protein sequ ence reveals that the 88-kDa polypeptide corresponds to a human homolo g of the Saccharomyces cerevisiae BRF protein, a subunit of yeast TFII IB. Human BRF (hBRF) probably corresponds to TFIIIB90, a protein previ ously cloned by Wang and Roeder (Proc. Natl, Acad. Sci, USA 92:7026-70 30, 1995), although its predicted amino acid sequence differs from tha t reported for TFIIIB90 over a stretch of 67 amino acids as a result o f frameshifts. Immunodepletion of more than 90 to 95% of the hBRF pres ent in a transcription extract severely debilitates transcription from the tRNA-type VAI promoter but does not affect transcription from the TATA box-containing human U6 promoter suggesting that the 0.38M-TFIII B complex, and perhaps hBRF as well, is not required far U6 transcript ion.