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，因为它们是从同一DNA链上的大簇（50-500 kb）相邻基因多顺反子转录的，成熟mRNA是通过反式剪接从初级转录物产生的。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观察到的调控，并且附加型分子被杂乱地转录。这导致了这样的假设，即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。然而，我们最近对L. major认为情况并非如此，并且在基因组中仅存在少量的特异性pol II启动子。然而，这些启动子似乎与典型的真核启动子显著不同，因为它们是双向的，缺乏TATA盒，并且具有多个转录起始位点（TIS）。此外，锥虫似乎缺乏，或具有高度不同的版本，大多数典型的真核细胞pol II转录因子。因此，我们假设，在这些生物体中，蛋白质编码基因的pol II转录是根本不同的。锥虫基因组计划的序列信息最近爆炸提供了一个很好的机会来表征整个染色体的转录组织和识别的转录机制。为了验证我们的假设，我们打算：1。描述了L.通过北方印迹和核连续分析的主要Friedlin（LmjF）; 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组分;以及3.使用体外和体内方法研究pol II机器和DNA模板之间的分子相互作用。这些研究旨在阐明LmjF中蛋白编码基因的pol II转录的分子过程。我们希望，他们将揭示锥虫和其他真核生物的转录过程之间的根本差异，这可能是有希望的开发新的化疗药物。

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.