Analysis of the yeast transcription factor TFIIA: distinct functional regions and a polymerase II-specific role in basal and activated transcription.
To probe the structure and function of the Saccharomyces cerevisiae general transcription factor TFIIA, we have systematically mutagenized the genes encoding both subunits and analyzed the effects of the mutations both in vivo and in vitro. We found that the central nonconserved region of the large subunit is not essential for function and likely acts as a spacer between the conserved N- and C-terminal regions. Deletion mutagenesis of the large subunit defined a region which is required for TATA binding protein (TBP) interaction. Alanine scanning mutagenesis defined a cluster of four basic residues which are likely required for interaction with DNA in the TBP-DNA complex. Much of the conserved regions of both subunits is required for subunit association, suggesting that these conserved regions fold into compact domains which extensively interact. In vitro transcription performed with extracts from yeast strains with mutations in either the large or the small TFIIA subunit demonstrated that TFIIA stimulates both basal and activated polymerase II (Pol II) transcription. The TFIIA-depleted extracts have normal Pol I and Pol III transcription activity, showing that TFIIA is a Pol II-specific factor. In vivo depletion of TFIIA activity reduced transcription from four different Pol II promoters. Finally, alanine scanning mutagenesis of TFIIA's small subunit has identified at least one mutation which is defective in transcription but which is not defective in subunit association or binding to TBP or TBP-DNA complexes.
Kang
J J
JJ
Basic Sciences Division, Fred Hutchinson Cancer Research Center, Seattle, Washington 98104.
Auble
D T
DT
Ranish
J A
JA
Hahn
S
S
D000409:Alanine; D000595:Amino Acid Sequence; D001483:Base Sequence; D002846:Chromatography, Affinity; D017124:Conserved Sequence; D017931:DNA Primers; D004268:DNA-Binding Proteins / Q000378:metabolism; D005800:Genes, Fungal; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D016296:Mutagenesis; D016297:Mutagenesis, Site-Directed; D016366:Open Reading Frames; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D012318:RNA Polymerase I / Q000378:metabolism; D012319:RNA Polymerase II / Q000378:metabolism*; D012320:RNA Polymerase III / Q000378:metabolism; D012333:RNA, Messenger / Q000096:biosynthesis; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D017384:Sequence Deletion; D016385:TATA Box; D035181:TATA-Box Binding Protein; D035561:Transcription Factor TFIIA; D014157:Transcription Factors / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D014158:Transcription, Genetic
D017931:DNA Primers; D004268:DNA-Binding Proteins; D046911:Macromolecular Substances; D012333:RNA, Messenger; D035181:TATA-Box Binding Protein; D035561:Transcription Factor TFIIA; D014157:Transcription Factors; D012319:RNA Polymerase II; D012318:RNA Polymerase I; D012320:RNA Polymerase III; D000409:Alanine