Structural studies on the Initiator protein, IBP39.

真核生物中蛋白质编码基因的启动子具有由核心启动子和 基因特异性调节剂的结合部位。转录起始是在核心启动子上介导的，它包括 转录起始点。尽管对真核转录进行了广泛的研究，但启动的机制细节 这一过程在很大程度上是未知的。更不清楚的是转录调控和启动的机制。 单细胞寄生原生动物。这些生物中的启动子似乎缺乏典型的后生动物核心启动子。 元素，如塔塔箱或发起人(INR)。然而，最近的研究表明，所有核心启动子在 阴道毛滴虫虽然不含TATA，但含有INR成分。阴道毛滴虫INR因子负责 与高等真核生物不同的是，在所有基因中，转录起始位置的选择只有大量蛋白质 在INR功能中，阴道毛滴虫/nr与一个39 kDa的“启动子结合蛋白”IBP39结合。IBP39， 它包含两个结构域，N-末端14.5 kDa的INR结合 结构域(IBD)通过蛋白水解物敏感连接到未知的C-末端24.5 kDa结构域(C-结构域) 功能。到目前为止，INR结合蛋白识别INR的结构基础，什么构成功能性INR， INR如何发挥调节转录起始的作用尚不清楚。因为阴道毛滴虫似乎有一种 简化的转录机制，仅依赖于INR，它为研究INR提供了一个很好的模型系统 通过结构/功能研究IBP39转录启动的结构基础及其与INR的相互作用。 我们最近对IBP39C结构域的测定和分析以及随后的生化研究证明 令人兴奋的发现是该结构域与阴道毛滴虫RNA聚合酶(RNAP)的C末端结构域(CTD)结合 U)大亚基，提示它可能具有将RNAP II招募到INR位点的功能。因此，这些研究已经 提供了对IBP39功能的关键初步见解。因此，为了提供对阴道毛滴虫INR的完整机制的理解 我们提出了以下目标：(1)确定IBD在存在和存在的情况下的晶体结构 不存在INR并通过荧光描绘构成功能性阴道毛滴虫INR的核苷酸元件 偏振(FP)实验。(2)确定IBP39C-结构域-RNAP II CTD复合体的结构。(3)至 C-结构域-CTD相互作用的生化表征及CTD磷酸化对此的影响 互动。IBP39在阴道毛滴虫所有蛋白编码基因的转录中所起的重要作用表明 是治疗滴虫病的一个可能的靶点，滴虫病每个人都有1.7亿人，

The promoters of protein-encoding genes in eukaryotes have a bipartite structure consisting of a core promoter and binding sites for gene specific regulators. Transcription initiation is mediated at the core promoter, which encompasses the transcription start site. Despite extensive studies on eukaryotic transcription, the mechanistic details of the initiation process are largely unknown. Even less well understood are the mechanisms of transcription regulation and initiation in single-celled parasitic protozoa. The promoters in these organisms appear to lack typical metazoan core promoter elements such as the TATA box or Initiator (Inr). However, recent studies have demonstrated that all core promoters in Trichomonas vaginalis, although TATA-less, contain Inr elements. The T. vaginalis Inr element is responsible for transcription start site selection in all genes and, unlike higher eukaryotes where numerous proteins have only been implicated in Inr function, the T. vaginalis/nr is bound by a single 39 kDa "initiator binding protein", IBP39. IBP39, which shows no sequence homology to any other protein, contains two domains, an N-terminal 14.5 kDa Inr binding domain (IBD) attached via a proteolyticaUy sensitive linker to a C-terminal 24.5 kDa domain (C-domain) of unknown function. To date, the structural basis for Inr recognition by an Inr-binding protein, what constitutes a functional Inr, and how the Inr functions to mediate transcription initiation remain unclear. Because T. vaginalis appears to have a simplified transcriptional mechanism, relying solely on the Inr, it provides an excellent model system for studying the structural basis of transcription initiation through structure/function studies on IBP39 and its interaction with the Inr. Our recent IBP39 C-domain structure determination and analysis and subsequent biochemical studies has demonstrated the exciting finding that this domain binds the C-terminal domain (CTD) of the T. vaginalis RNA polymerase (RNAP U) large subunit, suggesting it might function to recruit RNAP II to the Inr site. These studies have, therefore, provided key initial insight into IBP39 function. Thus, to provide a full mechanistic understanding of T. vaginalis Inr mediated transcription we propose the following aims: (1) determine the crystal structure of the IBD in the presence and absence of the Inr and delineate the nucleotide elements that constitute a functional T. vaginalis Inr via fluorescence polarization (FP) experiments. (2) determine the structure of the IBP39 C-domain-RNAP II CTD complex. (3) To biochemically characterize the C-domain-CTD interaction and to examine the effects of CTD phosphorylation on this interaction. The essential role that IBP39 plays in transcription of all protein-encoding genes in T. vaginalis suggests it is a possible target for the treatment of trichomoniasis, which afflicts over 170 million individuals each _,ear,