Multifunctional class I transcription in T. brucei

描述（由申请方提供）：原生生物寄生虫布氏锥虫可导致人类和家畜致死性疾病，并通过其采采蝇媒介传播。该病原体基因表达的一个关键策略是利用高活性的RNA聚合酶（pol）I转录编码变异表面糖蛋白（VSG）和原环素的基因。这是独一无二的，因为在真核生物中，RNA pol I专门转录核糖体DNA，而所有mRNA都由RNA pol II合成。这些糖蛋白对寄生虫来说是必需的，因为它们形成了保护性的细胞表面涂层。此外，血流形式的锥虫中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。RNA聚合酶I在T.布氏杆菌感染涉及在寄生虫的生命周期中将酶募集到四种结构不同的启动子类型，在血流形式中将酶浓缩在两个不同的核隔室中，以及VSG和原环素基因转录的生命周期依赖性调节。这种功能多样性预示T.与其宿主对应物相比，布氏RNA聚合酶I经历与更多种类的因子或因子结构域的必要相互作用。这项计划的长期目标是了解T.布氏杆菌，并确定必要的因素，因素域，或蛋白质-蛋白质相互作用，可能利用寄生虫控制。本研究的目的在于：1.前环和血流形式锥虫RNA pol I复合物的表征和纯化。与其他生物一样，RNA聚合酶I可以形成全酶，其纯化可以包括基础转录因子和其他转录调节蛋白。确定一个生命周期特定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表征。我们已经在RNA pol I第二大亚基的N-末端鉴定了这样的结构域。3.将遗传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.