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 transcription of the adenovirus type 2 VAI gene, which have been designated 0.38M-TFIIIB and 0.48M-TFIIIB. While 0.48M-TFIIIB has not been characterized, 0.38M-TFIIIB corresponds to a TBP-containing complex. We describe here the purification of this complex, which consists of TBP and a closely associated polypeptide of 88 kDa, and the isolation of a cDNA corresponding to the 88-kDa polypeptide. The predicted protein sequence reveals that the 88-kDa polypeptide corresponds to a human homolog of the Saccharomyces cerevisiae BRF protein, a subunit of yeast TFIIIB. Human BRF (hBRF) probably corresponds to TFIIIB90, a protein previously cloned by Wang and Roeder (Proc. Natl. Acad. Sci. USA 92:7026-7030, 1995), although its predicted amino acid sequence differs from that reported for TFIIIB90 over a stretch of 67 amino acids as a result of frameshifts. Immunodepletion of more than 90 to 95% of the hBRF present 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-TFIIIB complex, and perhaps hBRF as well, is not required for U6 transcription.
Mital
R
R
Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 11724, USA.
Kobayashi
R
R
Hernandez
N
N
D000260:Adenoviruses, Human / Q000235:genetics; D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D005786:Gene Expression Regulation; D005814:Genes, Viral; D006801:Humans; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic / Q000235:genetics; D012320:RNA Polymerase III / Q000235:genetics*; D012367:RNA, Viral / Q000235:genetics*; D017414:Ribonucleoprotein, U4-U6 Small Nuclear / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins; D016415:Sequence Alignment; D035381:TATA-Binding Protein Associated Factors; D035361:Transcription Factor TFIIIB; D014157:Transcription Factors / Q000235:genetics* / Q000302:isolation & purification; D014158:Transcription, Genetic
C090956:BRF1 protein, S cerevisiae; C441028:BRF1 protein, human; D012367:RNA, Viral; D017414:Ribonucleoprotein, U4-U6 Small Nuclear; D029701:Saccharomyces cerevisiae Proteins; D035381:TATA-Binding Protein Associated Factors; D035361:Transcription Factor TFIIIB; D014157:Transcription Factors; C052835:adenovirus associated RNA; D012320:RNA Polymerase III