Mechanism of Transcription Control by Drosophila TAF250

描述（由申请人提供）：TAFII250是TFIID中与TBP络合的10多种TBP结合蛋白相关因子（TAFIIs）之一。TFIID与基因转录起始位点周围的核心启动子结合，形成起始前复合物（pre - initiation complex, PlC）的装配核，内含RNA聚合酶II （Pol II）和一般转录因子TFIIA、TFIIB、TFIIE、TFIIF和TFIIH。TFIID的成核功能被认为包括几个不同的事件：(1)核心启动子DNA元件的激活子依赖性识别；(2)形成有利于PlC组装和转录起始的染色质环境；(3)对gtf进行结构和功能修饰，以促进PlC组装和转录起始。TAFII250在体外的活性与TAFII250在这些TFIID事件中的作用一致，但支持TAFII250在染色质背景下的这些或其他特定作用的证据不足。我们假设单个TAFII250活性在RNA Pol II转录过程中发挥基因特异性作用，信号通路和激活因子利用单个TAFII250活性来调节特定基因的转录。因此，在该基金的第1个目标中，我们将使用无偏遗传筛选来鉴定对果蝇生存能力重要的TAFII250结构域和残基。该筛选将使我们能够识别TAFII250的生物学相关特征。在本项目的第二部分，我们将确定TAFII250 c端区（CTR）异构体的差异表达或差异染色质关联是否有助于果蝇的基因特异性转录，以及在第二部分中鉴定的TAFII250特征是否有助于这一机制。在本研究的第3个目标中，我们将确定TAFII250 CTR异构体的差异生化活性是否有助于果蝇的基因特异性转录，以及第1个目标中确定的TAFII250特征是否有助于这一机制。确定个体TAFII250活性如何促进体内转录起始的一系列复杂的顺序相互作用和构象变化，将有助于更好地理解基因表达被调节的机制，以及该过程中的缺陷如何导致癌症和发育异常。

DESCRIPTION (provided by applicant): TAFII250 is one of more than 10 TATA-binding protein (TBP)-associated factors (TAFIIs) that are complexed with TBP in TFIID. Binding of TFIID to the core promoter surrounding the transcription start site of a gene nucleates assembly of the preinitiation complex (PlC), which contains RNA polymerase II (Pol II) and the general transcription factors TFIIA, TFIIB, TFIIE, TFIIF, and TFIIH. The nucleating function of TFIID is thought to comprise several distinct events: (1) activator-dependent recognition of core promoter DNA elements; (2) the generation of a chromatin environment that is favorable to PlC assembly and transcription initiation; and (3) structural and functional modification of GTFs to facilitate PlC assembly and transcription initiation. Activities attributed to TAFII250 in vitro are consistent with roles for TAFII250 in each of these TFIID events, but evidence supporting these or other specific roles for TAFII250 in the context of chromatin is insufficient. We hypothesize that individual TAFII250 activities play gene-specific roles during RNA Pol II transcription and that signaling pathways and activators utilize individual TAFII250 activities to modulate the transcription of specific genes. Thus, in Aim 1 of the grant, we will use an unbiased genetic screen to identify TAFII250 domains and residues that are important for Drosophila viability. This screen will allow us to identify biologically relevant features of TAFII250. In Aim 2 of the grant, we will determine whether differential expression or differential chromatin association of TAFII250 C-terminal region (CTR) isoforms contributes to gene-specific transcription in Drosophila and whether TAFII250 features identified in Aim 1 contribute to this mechanism. In Aim 3 of the grant, we will determine whether differential biochemical activities of TAFII250 CTR isoforms contribute to gene-specific transcription in Drosophila and whether TAFII250 features identified in Aim 1 contribute to this mechanism. The determination of how individual TAFII250 activities contribute to the complex series of sequential interactions and conformational changes that promote transcription initiation in vivo will lead to a better understanding of mechanisms through which gene expression is regulated and how defects in this process lead to cancer and developmental abnormalities.