Structure-function analysis of TAF130: identification and characterization of a high-affinity TATA-binding protein interaction domain in the N terminus of yeast TAF(II)130.

Bai Y, Perez GM, Beechem JM, Weil PA
Mol Cell Biol. 1997 17 (6): 3081-93

PMID: 9154807 · PMCID: PMC232161 · DOI:10.1128/mcb.17.6.3081

We report structure-function analyses of TAF130, the single-copy essential yeast gene encoding the 130,000-Mr yeast TATA-binding protein (TBP)-associated factor TAF(II)130 (yTAF(II)130). A systematic family of TAF130 mutants was generated, and these mutant TAF130 alleles were introduced into yeast in both single and multiple copies to test for their ability to complement a taf130delta null allele and support cell growth. All mutant proteins were stably expressed in vivo. The complementation tests indicated that a large portion (amino acids 208 to 303 as well as amino acids 367 to 1037) of yTAF(II)130 is required to support cell growth. Direct protein blotting and coimmunoprecipitation analyses showed that two N-terminal deletions which remove portions of yTAF(II)130 amino acids 2 to 115 dramatically decrease the ability of these mutant yTAF(II)130 proteins to bind TBP. Cells bearing either of these two TAF130 mutant alleles also exhibit a slow-growth phenotype. Consistent with these observations, overexpression of TBP can correct this growth deficiency as well as increase the amount of TBP interacting with yTAF(II)130 in vivo. Our results provide the first combined genetic and biochemical evidence that yTAF(II)130 binds to yeast TBP in vivo through yTAF(II)130 N-terminal sequences and that this binding is physiologically significant. By using fluorescence anisotropy spectroscopic binding measurements, the affinity of the interaction of TBP for the N-terminal TBP-binding domain of yTAF(II)130 was measured, and the Kd was found to be about 1 nM. Moreover, we found that the N-terminal domain of yTAF(II)130 actively dissociated TBP from TATA box-containing DNA.

MeSH Terms (21)

Amino Acid Sequence Binding Sites Conserved Sequence DNA-Binding Proteins Fluorescence Polarization Histone Acetyltransferases Molecular Sequence Data Nuclear Proteins Phenotype Saccharomyces cerevisiae Saccharomyces cerevisiae Proteins Sequence Deletion Structure-Activity Relationship TATA-Binding Protein Associated Factors TATA-Box Binding Protein TATA Box Trans-Activators Transcription, Genetic Transcription Factors Transcription Factors, TFII Transcription Factor TFIID

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