RIBOSOMAL RNA TRANSCRIPTION FACTOR FUNCTIONAL ANALYSIS

在真核细胞中，核糖体RNA基因的启动子 由几个区域组成：一个核心和不同数量的 上游元素。 核心的作用是将一个或多个 反式作用转录因子。 在阿米巴原虫中， 因子TIF与核心中的确定序列结合。 在这 在实验室中，TIF已被高度纯化，其相互作用 与DNA的突变已被详细表征， 启动子和足迹技术。 之间的相互作用 TIF和聚合酶也具有相同的特征。 方法. 其中重要的发现是，TIF指导 RNA聚合酶I结合转录起始位点， 蛋白质与蛋白质接触;聚合酶不产生序列特异性 在结合过程中与DNA接触。 此外，rRNA 转录受RNA聚合酶的修饰调节， 并且所述修饰防止聚合酶结合到所述聚合酶上， 提示可能影响TIF聚合酶的启动子 互动 为了更全面地了解 rRNA表达的起始和调控， TIF基因。 将对纯化的TIF进行部分测序 以及由预测的DNA合成的寡核苷酸探针 顺序 一个基因组文库，由于小的基因组， 阿米巴原虫的复杂性仅由9200个斑块组成， 进行TIF基因筛查。 测序后，基因组 克隆将用于筛选cDNA文库。 克隆的TIF基因 将在体外进行诱变、转录和翻译， 使用几种测定法鉴定蛋白质的功能结构域。 模板提交、足迹法和凝胶迁移试验将在 用于评估DNA结合。 转录径流测定和 DNA足迹法将用于鉴定TIF介导的 聚合酶结合和活化。 足迹分析将 确定突变TIF结合DNA的影响 与野生型因子相关的构象变化 约束力 从长远来看，TIF的互动细节 和聚合酶将使用位点特异性点 TIF基因和克隆的RNA聚合酶亚基的诱变 基因。

In eukaryotic cells, the promoter for the ribosomal RNA gene consists of several regions: a core and a varying number of upstream elements. The role of the core is to bind one or more trans-acting transcription factors. In Acanthamoeba, a single factor, TIF, binds to a defined sequence in the core. In this laboratory, TIF has been very highly purified, and its interaction with the DNA has been characterized in detail by mutagenesis of the promoter and footprinting techniques. Interactions between TIF and polymerase also have been characterized by the same methods. Among the significant findings are that TIF directs RNA polymerase I to bind over the transcription start site by protein-protein contact; polymerase makes no sequence-specific contacts with the DNA during binding. Further, rRNA transcription is regulated by modification of the RNA polymerase, and the modification prevents polymerase from binding to the promoter suggesting a possible effect on the TIF-polymerase interaction. In order to more fully understand the mechanism of initiation and regulation of rRNA expression, the cloning of the TIF gene is proposed. The purified TIF will be partially sequenced and an oligonucleotide probe synthesized from the predicted DNA sequence. A genomic library, which because of the small genomic complexity of Acanthamoeba consists of only 9200 plaques, will be screened for the TIF gene. Following sequencing, the genomic clone will be used to screen a cDNA library. The cloned TIF gene will be mutagenized, transcribed and translated in vitro, and the functional domains of the protein identified using several assays. Template commitment, footprinting, and gel shift assays will be used to evaluate DNA binding. Transcription run-off assays and DNA footprinting will be used to identify TIF-mediated polymerase binding and activation. Footprinting assays will determine the effects of mutant TIF binding on DNA conformational changes associated with the wild-type factor binding. In the long term, the details of the interaction of TIF and polymerase will be evaluated using site-specific point mutagenesis of the TIF gene and cloned RNA polymerase subunit gene(s).