RNA polymerase II transcription in trypanosomes

锥虫中的 RNA 聚合酶 II 转录

基本信息

批准号：
8827661
负责人：
ARTHUR GUNZL
金额：
$ 38.24万
依托单位：
UNIVERSITY OF CONNECTICUT SCH OF MED/DNT
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-05-15 至 2018-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8827661
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 Health High-Throughput Nucleotide Sequencing 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 research study transcription factor transcription factor TFIIE transcription factor TFIIF transcription factor TFIIH

项目摘要

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.

描述（由申请人提供）：Brucei，T。Cruzi和Leishmania spp的锥虫寄生虫锥虫。分别导致主要的人类疾病，分别导致非洲睡眠疾病，Chagas疾病和利什曼病。由于这些疾病的药物很少，有毒且难以施用，并且对这些药物的抗寄生虫具有抗药性，因此制定新的治疗策略变得越来越重要。尽管这些寄生虫已经开发出独特的宿主相互作用，但它们都具有相同的异常基因表达模式，涉及蛋白质编码基因的多余体转录和核前MRNA的反式剪接。在此过程中，一个关键分子是剪接的Leader（SL）RNA，将5'末端部分切割并融合到每个mRNA的5'端。由于在反剪接中消耗了SL RNA，因此寄生虫的生存力至关重要地取决于连续强的SL RNA RNA合成。因此，抑制SL RNA基因（SLRNA）转录似乎是针对锥虫寄生虫的有希望的宽带策略。到目前为止，我们已经能够识别和表征四个转录因子，其中包含T. Brucei中25种蛋白质的转录因子，这对于该过程至关重要：启动子结合复合物TRF4/SNAPC/TFIIA，TFIIB，TFIIB，TFIIH和TFIIE的复合体，以及Tripanosomys Mediator。我们的数据表明，这些蛋白质在SLRNA启动子上形成转录预发复合物，并募集RNA聚合酶II以进行准确的转录起始。相反，我们没有发现TFIIB与已知启动蛋白质编码基因阵列的RNA聚合酶II转录的DISVIIB结合。因此，我们建议在目标1中继续我们对RNA聚合酶II转录因子的生化表征，以确定最有希望的进一步分析目标。除指定的因素外，这些因素还包括与RNA POL II共纯化的新蛋白质以及新型TFIIA相关的复合物和与CDK相关的激酶，两者似乎都在MRNA合成中特别起作用。对于这些因素表征，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力纯化，条件基因沉默实验和体外转录测定。在AIM 2中，我们将使用一种系统的方法来研究DSSR中转录启动的机理，该机制将基于对RNA聚合酶II占用率的全基因组分析以及对DSSR驱动的报告基因表达的突变分析。总体而言，这些实验可能会在根本重要的过程中发现独特的和基本因素或因素域，即将RNA聚合酶II募集到DNA。此外，它们将对蛋白质表达的第一步提供机械理解，从而有助于控制锥虫。