Transcription of protein-coding genes in Leishmania

利什曼原虫蛋白质编码基因的转录

• 批准号: 7000361
• 负责人: Peter John Myler
• 金额: $ 37.3万
• 依托单位: SEATTLE BIOMEDICAL RESEARCH INSTITUTE
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-01-01 至 2007-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7000361
• 关键词: Transcription; protein; coding; genes; Leishmania

DESCRIPTION (provided by the applicant): Protein-coding genes in Leishmania and other related Trypanosomatidae are organized and transcribed in a unique fashion, in that they are transcribed polycistronically from large clusters (50-500 kb) of adjacent genes all on the same DNA strand, and the mature mRNAs are generated from the primary transcripts by trans-splicing. Moreover, while this transcription is carried out by RNA polymerase II (pol II), it appears to lack the regulation seen in other organisms, and episomal molecules are transcribed promiscuously. This has led to the hypothesis that pol II has very low specificity in these parasites, and that transcription can initiate indiscriminately throughout the genome. However, our recent transcriptional analysis of the complete chrl from L. major suggests that this is not the case, and that only a small number of specific pol II promoters are present in the genome. Nevertheless, it does appear that these promoters are significantly different from typical eukaryotic promoters, in that they are bi-directional, lack TATA boxes, and have multiple transcription initiation sites (TISs). In addition, the trypanosomatids appear to lack, or have highly divergent versions of, most of the typical eukaryotic pol II transcription factors. Thus, we hypothesize that pol II transcription of protein-coding genes is fundamentally different in these organisms. The recent explosion in sequence information from the trypanosomatid genome projects provides an excellent opportunity to characterize the transcriptional organization of entire chromosomes and identify the transcriptional machinery. In order to test our hypothesis, we intend to: 1. Characterize the transcriptional organization of several chromosomes of L. major Friedlin (LmjF) by Northern blot and nuclear run-on analyses; 2. Characterize the protein components of pol II transcriptional complexes in LmjF by bioinformatic and affinity purification/mass spectrometric approaches; and 3. Investigate the molecular interactions between the pol II machinery and DNA template using both in vitro and in vivo approaches. These studies are designed to elucidate the molecular processes underlying pol II transcription of protein-coding genes in LmjF. We expect that they will reveal fundamental differences between the transcriptional processes of trypanosomatids and other eukaryotes, which can hopefully be exploited for the development of novel chemotherapeutic agents.

描述（由申请人提供）：利什曼原虫和其他相关的锥虫科中的蛋白质编码基因以独特的方式进行组织和转录，因为它们是从大簇（50-500 kb）的邻近基因中抄录的，这些基因都是在相同的DNA链上产生的，并且是由同一DNA链上产生的，而成熟的MRNAS是由Trans-Splics trans-Splics s-Splics s-Splics s-Splics s-Splics splics splicsss。此外，尽管RNA聚合酶II（POL II）进行了这种转录，但似乎缺乏其他生物体中看到的调节，并且偶发分子被混合转录。这导致了以下假设：POL II在这些寄生虫中具有非常低的特异性，并且转录可以在整个基因组中不加选择地启动。但是，我们最近对L. Major的完整CHRL的转录分析表明，情况并非如此，并且基因组中只有少数特定的Pol II启动子。然而，这些启动子似乎与典型的真核启动子有显着不同的，因为它们是双向的，缺乏塔塔盒，并且具有多个转录启动位点（Tiss）。此外，大多数典型的真核Pol II转录因子似乎缺乏锥形剂，或具有高度不同的版本。因此，我们假设蛋白质编码基因的pol II转录在这些生物中根本不同。锥虫基因组项目的序列信息的最新爆炸爆炸为表征整个染色体的转录组织并识别转录机械提供了一个绝佳的机会。为了检验我们的假设，我们打算：1。通过Northern Blot和核跑步分析来表征弗里德林大杆菌（LMJF）的几种染色体的转录组织； 2。通过生物信息学和亲和力纯化/质谱方法来表征LMJF中POL II转录复合物的蛋白质成分；和3。使用体外和体内方法研究POL II机械和DNA模板之间的分子相互作用。这些研究旨在阐明LMJF中蛋白质编码基因的POL II转录的分子过程。我们预计它们将揭示锥形剂和其他真核生物的转录过程之间的根本差异，希望它们可以利用新型化学治疗剂的发展。