The conserved carboxy-terminal domain of Saccharomyces cerevisiae TFIID is sufficient to support normal cell growth.
We have examined the structure-function relationships of TFIID through in vivo complementation tests. A yeast strain was constructed which lacked the chromosomal copy of SPT15, the gene encoding TFIID, and was therefore dependent on a functional plasmid-borne wild-type copy of this gene for viability. By using the plasmid shuffle technique, the plasmid-borne wild-type TFIID gene was replaced with a family of plasmids containing a series of systematically mutated TFIID genes. These various forms of TFIID were expressed from three different promoter contexts of different strengths, and the ability of each mutant form of TFIID to complement our chromosomal TFIID null allele was assessed. We found that the first 61 amino acid residues of TFIID are totally dispensable for vegetative cell growth, since yeast strains containing this deleted form of TFIID grow at wild-type rates. Amino-terminally deleted TFIID was further shown to be able to function normally in vivo by virtue of its ability both to promote accurate transcription initiation from a large number of different genes and to interact efficiently with the Gal4 protein to activate transcription of GAL1 with essentially wild-type kinetics. Any deletion removing sequences from within the conserved carboxy-terminal region of S. cerevisiae TFIID was lethal. Further, the exact sequence of the conserved carboxy-terminal portion of the molecule is critical for function, since of several heterologous TFIID homologs tested, only the highly related Schizosaccharomyces pombe gene could complement our S. cerevisiae TFIID null mutant. Taken together, these data indicate that all important functional domains of TFIID appear to lie in its carboxy-terminal 179 amino acid residues. The significance of these findings regarding TFIID function are discussed.
Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, Tennessee 37232.
D
D
Poon
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S
Schroeder
C K
CK
Wang
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T
Yamamoto
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M
Horikoshi
R G
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Roeder
P A
PA
Weil
D000595:Amino Acid Sequence; D001483:Base Sequence; D004252:DNA Mutational Analysis; D005816:Genetic Complementation Test; D015151:Immunoblotting; D007700:Kinetics; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000254:growth & development; D013329:Structure-Activity Relationship; D035362:Transcription Factor TFIID; D014157:Transcription Factors / Q000235:genetics / Q000502:physiology*
D035362:Transcription Factor TFIID; D014157:Transcription Factors