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位点。转录起始在核心启动子处介导，其包括 转录起始位点。尽管对真核转录进行了广泛的研究，但启动的机制细节 过程基本上是未知的。更不清楚的是转录调节和启动的机制， 单细胞寄生原生动物。这些生物体中的启动子似乎缺乏典型的后生动物核心启动子 TATA盒或启动器（Inr）等元素。然而，最近的研究表明，所有的核心启动子， 阴道毛滴虫，虽然TATA少，含有Inr元素。霸王vagelinr元素负责 转录起始位点选择在所有基因中，不像高等真核生物，其中许多蛋白质只被 与Inr功能有关的T.迷走神经元/nr被单个39 kDa的“起始物结合蛋白”IBP 39结合。IBP 39， 其与任何其它蛋白质没有序列同源性，含有两个结构域，N-末端14.5kDa的Inr结合区， 通过蛋白水解敏感性接头连接至未知的C-末端24.5 kDa结构域（C-结构域）的IBD 功能迄今为止，Inr结合蛋白识别Inr的结构基础，即构成功能性Inr的物质， 以及Inr是如何介导转录起始的仍不清楚。因为T.流浪汉似乎有一个 简化的转录机制，仅依赖于Inr，它为研究 通过对IBP 39的结构/功能研究及其与Inr的相互作用，阐明了转录起始的结构基础。 我们最近的IBP 39 C-结构域结构测定和分析以及随后的生化研究表明， 令人兴奋的发现是，该结构域结合T.迷走神经RNA聚合酶 U）大亚基，这表明它可能具有将RNAP II募集到Inr位点的功能。因此，这些研究， 提供了对IBP 39功能的关键初步见解。因此，为了提供对T.迷走神经Inr 介导的转录，我们提出了以下目标：（1）确定IBD的晶体结构， Inr的缺失并描绘构成功能性T的核苷酸元件。荧光法测定迷走神经Inr 偏振（FP）实验。(2)确定IBP 39 C-结构域-RNAP II CTD复合物的结构。(3)到 生物化学表征C-结构域-CTD相互作用，并检查CTD磷酸化对此的影响 互动IBP 39在T.流浪汉说， 是治疗毛滴虫病的可能靶点，毛滴虫病每只耳朵折磨超过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,