Molecular mechanism of transactivation via transcriptional coactivator complexes

在真核基因表达中，包括RNA聚合酶（Pol） II在内的基础转录机制的募集是转录激活的主要机制。一个特定的转录因子与这个机制的成分相互作用，并将它们定位到一个特定的启动子上。如cAMP响应元件结合蛋白（CREB）通过募集CREB结合蛋白（CBP）刺激靶基因表达。CBP与磷酸化的CREB相互作用通过两种机制激活转录。一个依赖于组蛋白乙酰转移酶活性（HAT），另一个依赖于RNA解旋酶A （RHA）介导的Pol II募集。RHA是atp酶/解旋酶DExH家族的一员，可催化双链RNA和DNA从3‘到5’的位移。我们发现RHA将Pol II招募到乳腺癌特异性肿瘤抑制蛋白（BRCA1）中，并增强HIV病毒基因的表达。为了进一步了解RHA在基因表达中的作用，我们在本项目中确定了一个与Pol II相互作用的50个氨基酸的反活化结构域，并将其称为最小反活化结构域（MTAD）。该区域的蛋白质序列包含6个疏水残基，是RHA同源物所特有的，并且保守性很好。从全长RHA中删除该区域的突变体降低了creb依赖性转录的转录活性。此外，突变分析显示，MTAD中的几个色氨酸残基对于与Pol II的相互作用和交换活化是重要的。这些结果表明RHA可以通过募集Pol II或ATP依赖机制独立调节creb依赖的转录。

In the eukaryotic gene expression, recruitment of the basal transcriptional machinery including RNA polymerase (Pol) II is a principle mechanism for transcriptional activation. A specific transcriptional factor interacts with components of this machinery and localizes them to a specific promoter. For example, cAMP responsive element binding protein (CREB) stimulates target gene expression by recruitment of CREB binding protein (CBP). CBP interacting phosphorylated CREB activates the transcription via two mechanisms. One is dependent on the histone acetyltransferase activity (HAT), another is recruitment of Pol II mediated by RNA helicase A (RHA). RHA is a member of the DExH family of ATPases / helicases and catalyzes the displacement of both double-stranded RNA and DNA from 3' to 5'. We found that RHA recruits Pol II to breast cancer specific tumor suppressor protein (BRCA1) and enhance HIV virus gene expression.For further an understanding of the role of RHA on gene expression, we have identified a 50 amino acid transactivation domain that interacts with Pol II and termed it the minimal transactivation domain (MTAD) in this project. The protein sequence of this region contains six hydrophobic residues and is unique to RHA homologues and well conserved. A mutant with this region deleted from full-length RHA decreased transcriptional activity in CREB-dependent transcription. In addition, mutational analyses revealed that several tryptophan residues in MTAD are important for the interaction with Pol II and transactivation. These results suggest that RHA could independently regulate CREB-dependent transcription either through recruitment of Pol II or ATP dependent mechanisms.