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
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
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DOI：
10.1128/mcb.17.6.3081
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发表时间：
1997-06-01
影响因子：
5.3
通讯作者：
Weil, PA
中科院分区：
文献类型：
--
作者：
Bai, Y;Perez, GM;Weil, PA
我们报告了TAF 130的结构-功能分析，TAF 130是编码130，000-M-r酵母TATA结合蛋白（TBP）相关因子TAF（II）130（yTAF（II）130）的单拷贝必需酵母基因。产生TAF 130突变体的系统家族，并且将这些突变TAF 130等位基因以单拷贝和多拷贝引入酵母中以测试它们补充taf 130 Delta无效等位基因和支持细胞生长的能力。所有突变体蛋白在体内稳定表达。互补试验表明，支持细胞生长需要yTAF（II）130的大部分（氨基酸208至303以及氨基酸367至1037）。直接蛋白质印迹和共免疫沉淀分析表明，两个N-末端缺失，删除部分的yTAF（II）130氨基酸2至115显着降低这些突变的yTAF（II）130蛋白结合TBP的能力。携带这两种TAF 130突变等位基因的细胞也表现出缓慢生长表型。与这些观察结果一致，TBP的过表达可以纠正这种生长缺陷以及增加体内与yTAF（II）130相互作用的TBP的量。我们的研究结果提供了第一个综合的遗传和生物化学证据表明，yTAF（II）130结合酵母TBP在体内通过yTAF（II）130 N-末端序列，这种结合是生理上显着的。通过使用荧光各向异性光谱结合测量，测量了TBP与yTAF（II）130的N-末端TBP结合结构域的相互作用的亲和力，发现K-d约为1 nM。此外，我们发现yTAF（II）130的N-末端结构域主动地将TBP从含有TATA盒的DNA中解离。
We report structure-function analyses of TAF130, the single-copy essential yeast gene encoding the 130,000-M-r 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 taf130 Delta 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 K-d 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.