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: 6569306
• 负责人: Peter John Myler
• 金额: $ 35.8万
• 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/6569306
• 关键词: DNA directed RNA polymerase; DNA footprinting; Leishmania; Leishmania major; electrospray ionization mass spectrometry; gel mobility shift assay; genetic transcription; intermolecular interaction; liquid chromatography mass spectrometry; northern blottings; nuclear runoff assay; protozoal genetics; transfection

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锥虫科中的蛋白质编码基因以独特的方式组织和转录，因为它们是从相同 DNA 上的相邻基因的大簇（50-500 kb）多顺反子转录的链，成熟的 mRNA 是通过反式剪接从初级转录物生成的。此外，虽然这种转录是由 RNA 聚合酶 II (pol II) 进行的，但它似乎缺乏在其他生物体中看到的调节，并且附加型分子的转录是混杂的。这导致了这样的假设：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区别地启动。然而，我们最近对 L. Major 的完整 crl 进行的转录分析表明情况并非如此，并且基因组中仅存在少量特定的 pol II 启动子。然而，这些启动子确实与典型的真核启动子显着不同，因为它们是双向的，缺乏 TATA 盒，并且具有多个转录起始位点 (TIS)。此外，锥虫似乎缺乏大多数典型的真核 pol II 转录因子，或者具有高度不同的版本。因此，我们假设这些生物体中蛋白质编码基因的 pol II 转录存在根本不同。最近锥虫基因组计划中序列信息的爆炸性增长为表征整个染色体的转录组织和识别转录机制提供了绝佳的机会。为了检验我们的假设，我们打算： 1. 通过 Northern blot 和核连载分析来表征 L. Major Friedlin (LmjF) 的几个染色体的转录组织； 2. 通过生物信息学和亲和纯化/质谱方法表征LmjF中pol II转录复合物的蛋白质成分； 3. 使用体外和体内方法研究 pol II 机制和 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 pol II 转录的分子过程。我们期望它们将揭示锥虫和其他真核生物转录过程之间的根本差异，这有望用于开发新型化疗药物。