Trypanosome class II transcription pre-initiation complex

锥虫 II 类转录前起始复合物

基本信息

批准号：
7843594
负责人：
ARTHUR GUNZL
金额：
$ 38.25万
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 2011-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7843594
关键词：
African Trypanosomiasis Binding Biochemical Biological Assay Chagas Disease Cleaved cell Code Complex DNA DNA-Protein Interaction Disease Drug resistance Enzymes Eukaryota Exhibits Factor Analysis Failure Family Gene Expression General Transcription Factors Genes Genetic Transcription Genome Genomics Growth Homologous Gene Human In Vitro Insecta Intervention Leishmania Leishmaniasis Life Cycle Stages Maps Mastigophora Messenger RNA Mini-Exons Nature Nuclear Organism Parasites Pharmaceutical Preparations Plants Process Property Protein Array Protein Binding Proteins Prothrombin Public Health RNA RNA Interference RNA Polymerase II RNA chemical synthesis Recruitment Activity Role Sequence Analysis Site Spliced Leader RNA Spliced Leader Sequences Structure Technology Trans-Splicing Transcription Factor TFIIA Transcription Factor TFIIB Transcription Initiation Transcription Initiation Site Trypanosoma Trypanosoma brucei brucei Trypanosoma cruzi Trypanosomatina Work chromatin immunoprecipitation design human disease mRNA Precursor man novel therapeutics prevent promoter protein expression research study transcription factor transcription factor TFIIE transcription factor TFIIF transcription factor TFIIH

项目摘要

The trypanosomatid parasites Trypanosoma brucei, T. cruzi and Leishmania spp. (Tritryp) cause the major human diseases African Sleeping Sickness, Chagas’ disease, and Leishmaniasis, respectively. Drugs for these diseases are few and toxic and parasite resistance to these drugs is on the rise. Thus, it becomes increasingly important to find new anti-parasitic targets and develop new therapeutic strategies. All trypanosomatids depend on the same unusual mode of gene expression involving polycistronic transcription of protein coding genes and trans splicing of nuclear pre-mRNA. Of central importance to this process is the parasite-specific 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 this process, the parasites crucially depend on continuously strong SL RNA synthesis throughout their life cycle. We have recently identified and characterized several proteins in T. brucei which are essential for SL RNA gene transcription. These include extremely divergent homologues of the general transcription factors TFIIA, TFIIB, TFIIH, and a candidate TFIIE suggesting that the parasites form a class II transcription pre-initiation complex (PIC) at the SL RNA gene (SLRNA) promoter to recruit RNA polymerase II for accurate transcription initiation. The extraordinary level of sequence divergence between these proteins and their mammalian orthologues, which prevented their identification in the completed Tritryp genome projects, indicates that the trypanosome PIC deviates substantially from its human counterpart. To explore this possibility, we will functionally characterize the PIC which forms at the SLRNA promoter. This will include RNA interference, in vitro transcription experiments and DNA-protein interaction assays to evaluate the identified proteins’ importance for parasite growth and role in SLRNA transcription. Finally, we will analyze if the parasites use PIC formation to recruit RNA polymerase II to their long tandem arrays of protein coding genes. Chromatin immunoprecipitation in combination with deep sequencing will identify non-SLRNA PIC formation sites in the T. brucei genome. Together, these experiments may uncover unique and essential structural and/or biochemical features in a fundamentally important process, namely the recruitment of RNA polymerase II to DNA.

锥虫寄生虫布氏锥虫、克氏锥虫和利什曼原虫（Tritryp）分别引起人类主要疾病非洲昏睡病、南美锥虫病和利什曼病。治疗这些疾病的药物很少且有毒，并且寄生虫对这些药物具有抗药性。因此，寻找新的抗寄生虫靶标并开发新的治疗策略变得越来越重要。所有锥虫都依赖于相同的不寻常模式。基因表达的过程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，对此过程至关重要的是寄生虫特异性剪接前导 (SL) RNA，其中 5' 末端部分被切割并融合。由于 SL RNA 在此过程中被消耗，因此寄生虫在其整个生命周期中至关重要地依赖于持续强的 SL RNA 合成，我们最近在 T. brucei 中鉴定并表征了几种对于 T. brucei 至关重要的蛋白质。 SL RNA 基因转录。其中包括通用转录因子 TFIIA、TFIB、TFIIH 和候选 TFIIE 的极其不同的同源物，表明寄生虫在 SL RNA 基因 (SLRNA) 启动子处形成 II 类转录前起始复合物 (PIC)。招募 RNA 聚合酶 II 进行准确的转录起始。这些蛋白质与其哺乳动物直向同源物之间存在异常水平的序列差异，这阻碍了它们在已完成的 Tritryp 基因组计划中的识别，这表明锥虫 PIC 与其人类对应物有很大差异，为了探索这种可能性，我们将对 SLRNA 启动子处形成的 PIC 进行功能表征，这将包括 RNA 干扰、体外转录实验和 DNA-蛋白质相互作用测定，以评估已识别蛋白质的重要性。最后，我们将分析寄生虫是否利用 PIC 形成将 RNA 聚合酶 II 招募到其长串联蛋白质编码基因阵列中，并结合深度测序进行鉴定。总之，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）中独特且重要的结构和/或生化特征。