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首页> 外文期刊>Molecular and Cellular Biology >A new class of activation-defective TATA-binding protein mutants: evidence for two steps of transcriptional activation in vivo.
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A new class of activation-defective TATA-binding protein mutants: evidence for two steps of transcriptional activation in vivo.

机译：一类新的激活缺陷型TATA结合蛋白突变体：体内转录激活的两个步骤的证据。

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Using a genetic screen, we isolated four TATA-binding protein (TBP) mutants that are specifically defective in vivo for the response to acidic activators. In contrast to previously described activation-defective TBP mutants, these TBP derivatives are not specifically defective for interactions with TATA elements or TFIIA. Three of these derivatives interact normally with a TATA element, TFIIA, TFIIB, or an acidic activation domain; presumably, they affect another protein-protein interaction important for transcriptional activation. The remaining derivative (with F-237 replaced by D) binds a TATA element with wild-type affinity, but the TBP-TATA complex has an altered electrophoretic mobility and interacts poorly with TFIIA and TFIIB; this suggests that the conformation of the TBP-TATA element complex plays a role in transcriptional activation. To determine the step at which the TBP derivatives were unable to activate transcription, we utilized an artificial recruitment assay in which TBP is targeted to the promoter via fusion to the LexA DNA-binding domain. Consistent with previous evidence that acidic activators can increase recruitment of TBP to the promoter in vivo, the activation defect of some of these TBP derivatives can be corrected by artificial recruitment. In contrast, the activation defect of the other TBP derivatives is not bypassed by artificial recruitment. Thus, these TBP mutants define two steps in the process of transcriptional stimulation by acidic activators: efficient recruitment to the TATA element and a postrecruitment interaction with a component(s) of the initiation complex.
机译：使用遗传筛选，我们分离了四个TATA结合蛋白（TBP）突变体，这些突变体在体内对酸性激活剂的反应特别有缺陷。与先前描述的活化缺陷型TBP突变体相反，这些TBP衍生物对于与TATA元件或TFIAA的相互作用没有特别的缺陷。这些衍生物中的三个通常与TATA元件，TFIAA，TFIIB或酸性活化域相互作用。据推测，它们影响了对转录激活很重要的另一种蛋白质-蛋白质相互作用。剩余的衍生物（F-237被D取代）以野生型亲和力结合TATA元件，但是TBP-TATA复合物的电泳迁移率发生了变化，与TFIIA和TFIIB的相互作用较弱。这表明TBP-TATA元件复合物的构象在转录激活中起作用。为了确定TBP衍生物无法激活转录的步骤，我们利用了人工募集试验，其中TBP通过与LexA DNA结合域融合而靶向启动子。与先前的证据表明酸性激活剂可以在体内增加TBP向启动子的募集相一致，可以通过人工募集来纠正其中一些TBP衍生物的激活缺陷。相反，其他TBP衍生物的活化缺陷没有被人工募集绕过。因此，这些TBP突变体在酸性激活剂的转录刺激过程中定义了两个步骤：有效募集到TATA元件和招聘后与起始复合物的相互作用。

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