Mechanism of Transcription Control by Drosophila TAF250

描述(申请人提供)：TAFII250是TFIID中与TBP复合的10多个TATA结合蛋白相关因子(TAFIIs)之一。TFIID与基因转录起始点周围的核心启动子结合可使预起始复合体(PLC)组装成核，PLC包含RNA聚合酶II(Pol II)和通用转录因子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元件的依赖于激活剂的识别；(2)有利于PLC组装和转录启动的染色质环境的产生；(3)GTF的结构和功能修饰，以促进PLC组装和转录启动。TAFII250在体外的活性与TAFII250在这些TFIID事件中的角色是一致的，但支持TAFII250在染色质背景下的这些或其他特定角色的证据是不够的。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，信号通路和激活剂利用单个TAFII250活性来调节特定基因的转录。因此，在拨款的目标1中，我们将使用无偏见的遗传筛选来识别对果蝇生存至关重要的TAFII250结构域和残基。这一屏幕将允许我们识别TAFII250的生物相关特征。在拨款的目标2中，我们将确定TAFII250 C末端区域(CTR)亚型的差异表达或差异染色质结合是否有助于果蝇的基因特异性转录，以及在目标1中发现的TAFII250特征是否参与了这一机制。在拨款的目标3中，我们将确定TAFII250 CTR亚型的不同生化活性是否有助于果蝇的基因特异性转录，以及在目标1中发现的TAFII250特征是否有助于这一机制。确定单个TAFII250活性如何促进体内转录启动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的调控机制，以及这一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.