Transcription of protein-coding genes in Leishmania

描述（由申请人提供）：利什曼原虫和其他相关锥虫科的蛋白质编码基因以独特的方式组织和转录，因为它们是从同一DNA链上相邻基因的大簇（50-500 kb）中多顺反转录的，成熟的mrna是通过反式剪接从初级转录物中产生的。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控，并且附带体分子是混杂转录的。这导致了pol II在这些寄生虫中具有非常低特异性的假设，并且转录可以在整个基因组中不加选择地启动。然而，我们最近对L. major的完整chrl的转录分析表明情况并非如此，只有少数特定的pol II启动子存在于基因组中。然而，这些启动子似乎与典型的真核生物启动子有显著不同，因为它们是双向的，缺乏TATA盒，并且具有多个转录起始位点（TISs）。此外，锥虫似乎缺乏或具有大多数典型的真核pol II转录因子的高度不同的版本。因此，我们假设蛋白质编码基因的pol II转录在这些生物中是根本不同的。最近来自锥虫基因组计划的序列信息的爆炸提供了一个极好的机会来表征整个染色体的转录组织和鉴定转录机制。为了检验我们的假设，我们打算：1。利用Northern blot和核运行分析对L. major Friedlin （LmjF）染色体的转录组织进行了研究。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；和3。在体外和体内研究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中蛋白质编码基因的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，这有望用于开发新的化疗药物。

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.