Multifunctional class I transcription in T. brucei

描述(申请人提供)：布鲁氏锥虫原生寄生虫在人和牲畜中引起致命性疾病，并通过采采蝇媒介传播。利用高活性RNA聚合酶(PolI)转录编码变异表面糖蛋白(VSG)和原环素的基因是该病原菌基因表达的关键策略。这是独一无二的，因为在真核生物中，RNA polI只转录核糖体DNA，而所有的mRNA都是由RNA polII合成的。糖蛋白对寄生虫是必不可少的，因为它们形成了一层保护性的细胞表面涂层。此外，锥虫血液中VSG外壳的抗原变异是寄生虫逃避哺乳动物免疫系统的手段。在布氏毛滴虫中，RNA polI的多功能利用包括在寄生虫的生命周期中将该酶招募到四种结构不同的启动子类型，在血液形式的两个不同的核室中聚集该酶，以及依赖于生命周期对VSG和原环素基因转录的调节。这种功能多样性预示着布氏锥虫RNA polI与更多种类的因子或因子结构域发生了必要的相互作用。这一建议的长期目标是了解布鲁氏毛滴虫RNApolI转录的寄生虫特异性生物学，并确定可能用于寄生虫控制的基本因子、因子结构域或蛋白质-蛋白质相互作用。本研究的目的是：1.鉴定和纯化锥虫原环体和血流中的RNA polI复合体。与其他生物一样，RNA polI可能形成一个全酶，其纯化可能包括基本转录因子和其他转录调节蛋白。确定特定于生命周期的组成部分将是特别有意义的。2.寄生虫特异性转录因子或因子结构域的功能特征。我们已经在RNA polI第二大亚基的N端发现了这样一个结构域。3.将基因分析从启动子扩展到转录增强子和终止元件。4.纯化我们的体外转录活性，以便能够表征特定的DNA-蛋白质-相互作用，这可能有助于辅助转录因子的纯化和鉴定。

DESCRIPTION (provided by the applicant): The protist parasite Trypanosoma brucei causes lethal diseases in humans and livestock animals, and is transmitted by its tsetse vector. A key strategy in gene expression of this pathogen is to utilize highly active RNA polymerase (pol) I for the transcription of genes encoding variant surface glycoprotein (VSG) and procyclin. This is unique because in eukaryotes RNA pol I exclusively transcribe ribosomal DNA whereas all mRNA is synthesized by RNA pol II. The glycoproteins are essential for the parasite because they form a protective cell surface coat. Moreover, antigenic variation of the VSG coat in bloodstream form trypanosomes is the means by which the parasite evades the mammalian immune system. The multifunctional use of RNA pol I in T. brucei involves recruitment of the enzyme to four structurally different promoter types during the parasite's life cycle, concentration of the enzyme in two distinct nuclear compartments in bloodstream forms, and life cycle-dependent regulation of VSG and procyclin gene transcription. This functional diversity predicts that T. brucei RNA pol I undergoes essential interactions with a greater variety of factors or factor domains than its host counterparts. The long-term goal of this proposal is to understand the parasite-specific biology of RNA pol I transcription in T. brucei and to identify essential factors, factor domains, or protein-protein interactions which might be exploited for parasite control. The proposed study aims at: 1. Characterization and purification of RNA pol I complexes from both procyclic and bloodstream form trypanosomes. As in other organisms, RNA pol I may form a holoenzyme whose purification may include basal transcription factors and other transcription regulatory proteins. It will be of particular interest to identify a life cycle-specific component. 2. Functional characterization of parasite-specific transcription factors or factor domains. We have already identified such a domain at the N-terminus of the RNA pol I second largest subunit. 3. Extending the genetic analysis from promoters to transcription enhancer and termination elements. 4. Purification of our in vitro transcription activity to enable characterization of specific DNA-protein-interactions, which may facilitate purification and identification of auxiliary transcription factors.