Trypanosome class II transcription pre-initiation complex

锥虫 II 类转录前起始复合物

• 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
• 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

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）分别导致人类主要疾病非洲昏睡病、恰加斯病和利什曼病。治疗这些疾病的药物很少且有毒，而且寄生虫对这些药物的耐药性正在上升。因此，寻找新的抗寄生虫靶点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。此过程中最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到每个 mRNA 的 5' 末端。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大的 SL RNA 合成。我们最近在 T. brucei 中鉴定并表征了几种对于 SL RNA 基因转录至关重要的蛋白质。其中包括一般转录因子 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)，以招募 RNA 聚合酶 II 进行准确的转录起始。这些蛋白质和它们的哺乳动物直系同源物之间的序列差异非常大，这阻碍了它们在已完成的 Tritryp 基因组计划中的鉴定，表明锥虫 PIC 与其人类对应物有很大的不同。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对寄生虫生长的重要性以及在 SLRNA 转录中的作用。最后，我们将分析寄生虫是否利用 PIC 形成将 RNA 聚合酶 II 招募到其蛋白质编码基因的长串联阵列中。染色质免疫沉淀结合深度测序将鉴定 T. brucei 基因组中的非 SLRNA PIC 形成位点。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。