RNA polymerase II transcription in trypanosomes

描述（由申请方提供）：锥虫寄生虫布氏锥虫、T.克氏利什曼原虫属（Leishmania spp.）分别引起非洲昏睡病、恰加斯病和利什曼病等人类主要疾病。由于治疗这些疾病的药物很少，有毒且难以管理，寄生虫对这些药物的耐药性正在上升，因此开发新的治疗策略变得越来越重要。虽然这些寄生虫已经开发出独特的宿主-寄生虫相互作用，但它们都具有相同的不寻常的基因表达模式，涉及蛋白质编码基因的多顺反子转录和核前mRNA的反式剪接。这个过程中的关键分子是剪接前导（SL）RNA，其5'末端部分被切割并融合到每个mRNA的5'末端。由于SL RNA在反式剪接中被消耗，寄生虫的生存能力关键取决于持续强大的SL RNA合成。因此，抑制SLRNA基因（SLRNA）的转录似乎是一个很有前途的宽带策略，对锥虫寄生虫。到目前为止，我们已经能够确定和表征四个转录因子，包括25个蛋白质在T。这些蛋白质包括：启动子结合复合物TRF 4/SNAPc/TFIIA、TFIIB、TFIIH和TFIIE的复合物以及锥虫介体。我们的数据表明，这些蛋白质在SLRNA启动子处形成转录前起始复合物，并招募RNA聚合酶II用于准确的转录起始。相反，我们没有发现TFIIB结合到已知启动RNA聚合酶II转录的蛋白质编码基因阵列的发散链转换区（dSSR）的证据。因此，我们在目标1中提出继续我们的RNA聚合酶II转录因子的生物化学表征，以确定最有前途的目标，以进一步分析;除了指定的因素，这些包括新的蛋白质，共纯化与RNA聚合酶II以及一种新的TFIIA相关的复合物和CDK相关的激酶，这两个似乎特异性地在前mRNA合成中发挥作用。对于这些因素的表征，我们计划采用大量的遗传和生物化学实验，其中包括串联亲和纯化的蛋白质复合物，条件基因沉默实验和体外转录测定。在目标2中，我们将使用一种系统的方法来研究dSSR中的转录起始机制，该方法将基于RNA聚合酶II占用的全基因组分析和dSSR驱动的报告基因表达的突变分析。总的来说，这些实验可能会发现一个根本重要的过程中，即RNA聚合酶II的招聘DNA的独特和必要的因素或因素域。此外，它们将提供蛋白质表达的第一步的机制理解，这将有助于长期控制锥虫。

DESCRIPTION (provided by applicant): The trypanosomatid parasites Trypanosoma brucei, T. cruzi and Leishmania spp. cause the major human diseases African Sleeping Sickness, Chagas' disease, and Leishmaniasis, respectively. Since drugs for these diseases are few, toxic and difficult to administer, and parasite resistance to these drugs is on the rise, it becomes increasingly important to develop new therapeutic strategies. While these parasites have developed unique host-parasite interactions, they all share the same unusual mode of gene expression involving polycistronic transcription of protein coding genes and trans splicing of nuclear pre-mRNA. A key molecule in this process is the spliced leader (SL) RNA from which the 5' terminal part is cleaved and fused to the 5' end of each mRNA. Since SL RNA is consumed in trans splicing, parasite viability crucially depends on continuously strong SL RNA synthesis. Therefore, inhibition of SL RNA gene (SLRNA) transcription appears to be a promising broadband strategy against trypanosomatid parasites. So far, we have been able to identify and characterize four transcription factors comprising 25 proteins in T. brucei that are essential for the process: the promoter-binding complex TRF4/SNAPc/TFIIA, TFIIB, a complex of TFIIH and TFIIE, and the trypanosome mediator. Our data show that these proteins form a transcription pre-initiation complex at the SLRNA promoter and recruit RNA polymerase II for accurate transcription initiation. Conversely, we found no evidence that TFIIB binds to divergent strand switch regions (dSSRs) known to initiate RNA polymerase II transcription of the protein coding gene arrays. We therefore propose in Aim 1 to continue our biochemical characterization of RNA polymerase II transcription factors to identify the most promising targets for further analysis; besides the specified factors, these include new proteins that co-purified with RNA pol II as well as a novel TFIIA- associated complex and a CDK-related kinase both of which appear to function specifically in pre-mRNA synthesis. For these factor characterizations we plan to employ a plethora of genetic and biochemical experiments which include tandem affinity purification of protein complexes, conditional gene silencing experiments and in vitro transcription assays. In Aim 2, we will use a systematic approach to investigate the mechanism of transcription initiation in dSSRs which will be based on a genome-wide analysis of RNA polymerase II occupancy and on a mutational analysis of dSSR-driven reporter gene expression. Overall, these experiments may uncover unique and essential factors or factor domains in a fundamentally important process, namely the recruitment of RNA polymerase II to DNA. Moreover, they will provide a mechanistic understanding of the first step in protein expression which will help to control trypanosomatids in the long term.