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

We report structure-function analyses of TAF130, the single-copy essen tial yeast gene encoding the 130,000-M-r yeast TATA-binding protein (T BP)-associated factor TAF(II)130 (yTAF(II)130). A systematic family of TAF130 mutants was generated, and these mutant TAF130 alleles were in troduced into yeast in both single and multiple copies to test for the ir ability to complement a taf130 Delta null allele and support cell g rowth. All mutant proteins were stably expressed in vivo. The compleme ntation tests indicated that a large portion (amino acids 208 to 303 a s well as amino acids 367 to 1037) of yTAF(II)130 is required to suppo rt cell growth. Direct protein blotting and coimmunoprecipitation anal yses showed that two N-terminal deletions which remove portions of yTA F(II)130 amino acids 2 to 115 dramatically decrease the ability of the se mutant yTAF(II)130 proteins to bind TBP. Cells bearing either of th ese two TAF130 mutant alleles also exhibit a slow-growth phenotype. Co nsistent with these observations, overexpression of TBP can correct th is growth deficiency as well as increase the amount of TBP interacting with yTAF(II)130 in vivo. Our results provide the first combined gene tic and biochemical evidence that yTAF(II)130 binds to yeast TBP in vi vo through yTAF(II)130 N-terminal sequences and that this binding is p hysiologically significant. By using fluorescence anisotropy spectrosc opic binding measurements, the affinity of the interaction of TBP for the N-terminal TBP-binding domain of yTAF(II)130 was measured, and the K-d was found to be about 1 nM. Moreover, we found that the N-termina l domain of yTAF(II)130 actively dissociated TBP from TATA box-contain ing DNA.