Molecular cloning of the small (gamma) subunit of human TFIIA reveals functions critical for activated transcription.
TFIIA is thought to play an important role in transcriptional regulation in higher eukaryotes, but its precise function is unclear. A human cDNA encoding a protein with 45% identity to the small subunit of yeast TFIIA has been isolated. TFIIA activity could be reconstituted by the mixing of recombinant large (alpha beta) and small (gamma) subunits. TFIIA-depleted HeLa nuclear extracts were used to demonstrate that TFIIA is essential for basal and activated transcription by several distinct classes of activators. Recombinant TFIIA functioned in transcriptional activation whether expressed as a dimer (alpha beta+gamma) or as a trimer (alpha+beta+gamma), which closely resembles the native form. Yeast TFIIA also functioned in transcriptional activation, and the human gamma subunit was functionally interchangeable with TOA2, its yeast homolog. Recombinant TFIIA mediated the stimulation of TFIID binding to the TATA region and downstream promoter sequences by the Zta transcriptional activator. Significantly, we found that TFIIA bound directly to Zta in an activation domain-dependent manner. One consequence of the TFIIA-mediated interaction between Zta and TFIID was the formation of a promoter-bound complex resistant to TATA oligonucleotide competition. These results demonstrate that TFIIA is an evolutionarily conserved general factor critical for activator-regulated transcription.
Roche Institute of Molecular Biology, Nutley, New Jersey 07110-1199.
J
J
Ozer
P A
PA
Moore
A H
AH
Bolden
A
A
Lee
C A
CA
Rosen
P M
PM
Lieberman
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D017124:Conserved Sequence; D018076:DNA, Complementary / Q000235:genetics / Q000302:isolation & purification; D004268:DNA-Binding Proteins / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D016385:TATA Box; D035181:TATA-Box Binding Protein; D015534:Trans-Activators / Q000378:metabolism; D035561:Transcription Factor TFIIA; D035362:Transcription Factor TFIID; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D015533:Transcriptional Activation; D014764:Viral Proteins
C063676:BZLF1 protein, Herpesvirus 4, Human; D018076:DNA, Complementary; D004268:DNA-Binding Proteins; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D035181:TATA-Box Binding Protein; C441520:TOA2 protein, S cerevisiae; D015534:Trans-Activators; D035561:Transcription Factor TFIIA; D035362:Transcription Factor TFIID; D014157:Transcription Factors; D014764:Viral Proteins