RNA polymerase II transcription in trypanosomes

锥虫中的 RNA 聚合酶 II 转录

• 批准号: 8259403
• 负责人: ARTHUR GUNZL
• 金额: $ 38.26万
• 依托单位: UNIVERSITY OF CONNECTICUT SCH OF MED/DNT
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-05-15 至 2016-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8259403
• 关键词: Affinity Chromatography; African Trypanosomiasis; Binding; Binding Sites; Biochemical; Biological Assay; Cell physiology; Chagas Disease; Characteristics; Chromosomes, Human, Pair 3; Chromosomes, Human, Pair 9; Cleaved cell; Code; Complex; Cyclin-Dependent Kinases; DNA; DNA-Directed RNA Polymerase; Data; Development; Disease; Drug resistance; Electron Microscopy; Enzymes; Eukaryota; Exhibits; Gene Expression; Gene Silencing; General Transcription Factors; Genes; Genetic; Genetic Transcription; Genome; Homologous Gene; Human; In Vitro; Individual; Intervention; Investigation; Leishmania; Leishmania major; Leishmaniasis; Maps; Mediator of activation protein; Messenger RNA; Molecular; Morphology; Nuclear; Parasite Control; Parasite resistance; Parasites; Pharmaceutical Preparations; Phosphorylation; Phosphotransferases; Polyadenylation; Process; Proteins; RNA Polymerase II; RNA chemical synthesis; Recruitment Activity; Reporter; Reporter Genes; Research; Role; Site; Small RNA; Specific qualifier value; Spliced Leader RNA; Spliced Leader Sequences; System; TATA-Box Binding Protein; Technology; Testing; Trans-Splicing; Transcription Factor TFIIA; Transcription Factor TFIIB; Transcription Initiation; Transcription Initiation Site; Trypanosoma; Trypanosoma brucei brucei; Trypanosoma cruzi; Work; Yeasts; base; chromatin immunoprecipitation; genome-wide; genome-wide analysis; helicase; histone modification; human disease; insight; killings; mRNA Precursor; member; novel; novel therapeutics; particle; pathogen; promoter; protein complex; protein expression; protein purification; public health relevance; research study; transcription factor; transcription factor TFIIE; transcription factor TFIIF; transcription factor TFIIH

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. PUBLIC HEALTH RELEVANCE: The proposed studies of this application will explore mechanism and relevant factors of RNA polymerase II transcription in Trypanosoma brucei, a member of a group of parasites that cause major, devastating diseases in humans. The proteins involved in this process exhibit an unprecedented divergence level to their human counterparts which raises the possibility that we can identify new targets for chemotherapeutic intervention of the parasites. Such targets are urgently needed because drugs for this disease are few and toxic, and parasite resistance to existing drugs is on the rise. Moreover, a thorough understanding of trypanosome gene expression will help to control the parasite in the long term.

描述（由申请人提供）：锥虫寄生虫布氏锥虫、克氏锥虫和利什曼原虫。分别导致人类主要疾病非洲昏睡病、恰加斯病和利什曼病。由于治疗这些疾病的药物数量少、有毒且难以施用，并且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发展出独特的宿主-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。此过程中的一个关键分子是剪接前导 (SL) RNA，其中 5' 末端部分被切割并融合到每个 mRNA 的 5' 末端。由于 SL RNA 在反式剪接过程中被消耗，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 SL RNA 基因 (SLRNA) 转录似乎是对抗锥虫寄生虫的一种有前景的宽带策略。到目前为止，我们已经能够鉴定和表征布氏锥虫中包含 25 种蛋白的四种转录因子，这些转录因子对于该过程至关重要：启动子结合复合物 TRF4/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 以实现准确的转录起始。相反，我们没有发现任何证据表明 TFIIB 与已知可启动蛋白质编码基因阵列的 RNA 聚合酶 II 转录的不同链转换区 (dSSR) 结合。因此，我们在目标 1 中建议继续对 RNA 聚合酶 II 转录因子进行生化表征，以确定最有希望的目标进行进一步分析；除了特定的因素外，这些因素还包括与 RNA pol II 共纯化的新蛋白质以及新型 TFIIA 相关复合物和 CDK 相关激酶，这两者似乎在前 mRNA 合成中具有特异性功能。对于这些因子表征，我们计划采用大量的遗传和生化实验，其中包括蛋白质复合物的串联亲和纯化、条件基因沉默实验和体外转录测定。在目标 2 中，我们将基于对 RNA 聚合酶 II 占用的全基因组分析和对 dSSR 驱动的报告基因表达的突变分析，使用系统方法来研究 dSSR 中的转录起始机制。总体而言，这些实验可能会揭示一个极其重要的过程（即 RNA 聚合酶 II 向 DNA 的募集）中独特且重要的因子或因子域。此外，他们还将提供对蛋白质表达第一步的机制理解，这将有助于长期控制锥虫。 公共健康相关性：本申请的拟议研究将探索布氏锥虫（Trypanosoma brucei）中 RNA 聚合酶 II 转录的机制和相关因素，布氏锥虫是引起人类重大毁灭性疾病的寄生虫群体的成员。参与这一过程的蛋白质与人类对应的蛋白质表现出前所未有的差异水平，这提高了我们确定寄生虫化疗干预新靶点的可能性。迫切需要这样的目标，因为治疗这种疾病的药物很少且有毒，而且寄生虫对现有药物的耐药性正在上升。此外，彻底了解锥虫基因表达将有助于长期控制寄生虫。