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: 7160552
• 负责人: Peter John Myler
• 金额: $ 36.22万
• 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 2009-05-14
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7160552
• 关键词: Affinity Chromatography; Amino Acid Sequence; Antibodies; Binding; Bioinformatics; Biological Assay; Blast Cell; Chromosomes; Chromosomes, Human, Pair 1; Code; Complex; DNA; DNA Sequence; Databases; Deoxyribonuclease I; Deoxyribonucleases; Development; Enzymes; Eukaryota; Eukaryotic Cell; Explosion; General Transcription Factors; Genes; Genetic Transcription; Genome; Genomics; Histones; Homologous Gene; In Vitro; Label; Leishmania; Liquid Chromatography; Mediator of activation protein; Methods; Molecular; Northern Blotting; Nuclear; Nucleic Acids; Numbers; Organism; Parasites; Pattern; Polymerase; Process; Proteins; RNA; RNA Polymerase II; Regulation; Reporter; Research Design; Research Personnel; Running; Site; Specificity; Spectrometry, Mass, Electrospray Ionization; Staging; System; TATA Box; Testing; Trans-Splicing; Transcript; Transcription Initiation; Transcription Initiation Site; Transcriptional Regulation; Transfection; Trypanosoma; Trypanosoma brucei brucei; Trypanosoma cruzi; Trypanosomatina; Yeasts; chemotherapeutic agent; design; gene replacement; histone acetyltransferase; in vivo; novel; programs; promoter; protein reconstitution; research study; transcription factor

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.

利什曼原虫和其他相关的锥虫科中的蛋白质编码基因被组织和转录 独特的时尚，因为它们是从相邻基因的大簇（50-500 kb）中抄录的 所有在同一DNA链上都 剪接。此外，虽然RNA聚合酶II（POL II）进行了此转录，但似乎缺乏 在其他生物体中看到的调节和偶发分子被串通转录。这已经引起了 假设Pol II在这些寄生虫中的特异性非常低，并且该转录可以启动 在整个基因组中不加区分。但是，我们最近对完整CHL的转录分析 从L. Major提出的情况并非 存在于基因组中。然而，这些发起人似乎确实与 典型的真核启动子，因为它们是双向的，缺少塔塔盒，并且有多个 转录启动位点（Tiss）。此外，锥形剂似乎缺乏或具有高度分歧 大多数典型的真核Pol II转录因子的版本。因此，我们假设Pol II 在这些生物体中，蛋白质编码基因的转录从根本上有所不同。最近的爆炸 来自锥虫基因组项目的序列信息提供了一个绝佳的机会 表征整个染色体的转录组织并识别转录 机械。为了检验我们的假设，我们打算：1。 Northern印迹和核跑步分析的弗里德林大杆菌（LMJF）的几个染色体； 2。 表征生物信息学和亲和力在LMJF中POL II转录复合物的蛋白质成分 纯化/质谱方法；和3。研究Pol II之间的分子相互作用 使用体外和体内方法的机械和DNA模板。这些研究被设计为 阐明了LMJF中蛋白质编码基因的POL II转录的分子过程。我们期望 他们将揭示锥虫的转录过程和 其他真核生物有望被利用为开发新型化学治疗剂。