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锥虫科的蛋白质编码基因以 独特的方式，因为它们是从相邻基因的大簇（50-500 kb）多顺反子转录的 全部都在同一条 DNA 链上，并且成熟的 mRNA 是通过反式从初级转录物生成的 拼接。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，游离型分子的转录是混杂的。这导致了 假设 pol II 在这些寄生虫中具有非常低的特异性，并且转录可以启动 不加区别地分布在整个基因组中。然而，我们最近对完整 crl 的转录分析 来自 L. Major 表明情况并非如此，并且只有少数特定的 pol II 启动子是 存在于基因组中。然而，这些启动子确实与 典型的真核启动子，因为它们是双向的，缺乏 TATA 框，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分歧 大多数典型真核 pol II 转录因子的版本。因此，我们假设 pol II 这些生物体中蛋白质编码基因的转录有根本的不同。最近发生的爆炸 来自锥虫基因组计划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. 表征 通过 Northern 印迹和核连续分析，确定 L. Major Friedlin (LmjF) 的几条染色体； 2. 通过生物信息学和亲和力表征 LmjF 中 pol II 转录复合物的蛋白质成分 纯化/质谱方法； 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 他们将揭示锥虫和锥虫转录过程之间的根本差异 其他真核生物，有望用于开发新型化疗剂。