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。在棘阿米巴，一种单一的 因子，TIF，与核心中定义的序列结合。在这 实验室，TIF已经得到了非常高的提纯，并且它的相互作用 已经通过对DNA的诱变详细地表征了 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：研究方法。其中重要的发现是，TIF指导 RNA聚合酶I通过以下方式与转录起始点结合 蛋白质-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 转录是通过RNA聚合酶的修饰来调节的， 并且这种修饰阻止了聚合酶与 启动子提示对TIF-聚合酶的可能影响 互动。为了更全面地了解其作用机制。 RRNA表达的启动和调控，rRNA的克隆 提出了TIF基因。纯化的TIF将被部分测序 以及从预测的DNA合成的寡核苷酸探针 序列。一个基因组文库，因为基因组很小 棘阿米巴的复杂性只有9200个斑块，将 进行TIF基因筛查。测序后，基因组 克隆将被用来筛选cDNA文库。TIF基因的克隆 将在体外被诱变、转录和翻译，而 用几种分析方法鉴定了该蛋白质的功能结构域。 模板承诺、足迹和凝胶移动分析将是 用于评估DNA结合情况。转录径流分析和 DNA足迹将用于识别TIF介导的 聚合酶结合和激活。足迹分析将 检测突变型TIF结合对DNA的影响 与野生型因子相关的构象变化 有约束力的。从长远来看，TIF相互作用的细节 聚合酶将使用特定的位点进行评估 TIF基因的诱变及其RNA聚合酶亚基的克隆 基因(S)。

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).