TRANSCRIPTIONAL CONTROL OF MITOCHONDRIAL GENE EXPRESSION IN TRYPANOSOMES

摘要 布鲁氏锥虫原虫引起非洲人和动物锥虫病，一系列疾病 影响撒哈拉以南非洲的人口和经济。这些异源血鞭毛虫属于 动丝泡纲：一个分类纲，以拥有动丝泡体为特征。这个核蛋白小体含有 线粒体DNA(KDNA)：编码rRNA、核糖体蛋白和核糖体蛋白的~25个最大循环(每个~23kb) 呼吸复合体的亚基，以及大约5000个带有引导RNA基因的~1kb小环。 松弛的大环和小环相互关联，被包装成密集的盘状网络 含有类似组蛋白的蛋白质。几十年的kDNA研究揭开了一般生物学的迷人现象 意义，如DNA弯曲和mRNA编辑，并揭示了复制和RNA的精致细节 正在处理。然而，转录的分子机制实际上仍然没有被探索过，而且是有争议的。 构成了理解线粒体基因表达的最关键的空白。历久弥新的观点 多顺反子RNA合成的研究减少了研究转录对调控的贡献 基因组活动。相反，这一提议提供了上环和微环基因分别存在的证据。 转录成3‘端延伸的前体。转录起始点定义了前mRNA5‘末端，即 随后被焦磷水解酶复合体转化为单磷酸化状态，称为 PPome。大多数引导RNA缺乏PPome识别位点，并保持三磷酸化状态。此外，我们 建立反义转录本通过阻止成熟RNA的3‘-5’降解来划定成熟RNA的3‘端边界 3‘端过程组(MPome)的前体。由此推断，转录的起始点在正义和反义上 链分别定义5‘和3’的信使核糖核酸末端。这些发现支持线粒体基因的概念-- 在寄生虫的发育和发病机制中具有广泛意义的特异性转录调控。我们假设 阐明转录复合体的组成、DNA模板的要求和特定因子的功能 为这一新兴的研究领域奠定基础。我们建议：1)表征RNA聚合酶复合体 从血液和昆虫寄生虫的形式，并评估转录因子对RNA合成的贡献；2) MAP最大环和小环启动子；3)重组活性转录复合体。

ABSTRACT Parasitic protist Trypanosoma brucei causes African human and animal trypanosomiasis, a spectrum of diseases affecting the population and economy in sub-Saharan Africa. These digenetic hemoflagellates belong to Kinetoplastea, a taxonomic class distinguished by possession of a kinetoplast. This nucleoprotein body contains mitochondrial DNA (kDNA) of two kinds: ~25 maxicircles (each ~23 kb) encoding rRNAs, ribosomal proteins and subunits of respiratory complexes, and approximately 5000 of ~1 kb minicircles bearing guide RNA genes. Relaxed maxicircles and minicircles are interlinked and packed into a dense disc-shaped network by association with histone-like proteins. Decades of kDNA studies have unraveled fascinating phenomena of general biological significance, such as DNA bending and mRNA editing, and revealed exquisite details of replication and RNA processing. However, the molecular mechanisms of transcription remain virtually unexplored and arguably constitute the most critical gap in understanding mitochondrial gene expression. The historically enduring view of polycistronic RNA synthesis has abridged efforts to investigate transcription's contribution to regulating genome activity. In contrast, this proposal presents evidence that maxicircle and minicircle genes are individually transcribed into 3′ extended precursors. The transcription start site defines pre-mRNA 5′ terminus, which is subsequently converted into monophosphorylated state by a pyrophosphohydrolase complex, termed the PPsome. Most guide RNAs lack PPsome recognition sites and remain triphosphorylated. Furthermore, we establish that antisense transcripts delimit the 3′ boundaries of mature RNAs by blocking 3′-5′ degradation of precursors by the 3′ processome (MPsome). It follows that transcription start sites on sense and antisense strands define 5′ and 3′ mRNA termini, respectively. These findings support a concept of mitochondrial gene- specific transcriptional control with broad implications in parasite development and pathogenesis. We posit that elucidating transcription complex composition, DNA template requirements and functions of specific factors will build a foundation for this nascent research area. We propose to: 1) Characterize RNA polymerase complex from bloodstream and insect parasite forms, and assess transcription factors' contributions to RNA synthesis; 2) Map maxicircle and minicircle promoters; and 3) Reconstitute the active transcription complex.