TRANSCRIPTION OF ACANTHAMOEBA RNA POLYMERASE II GENES

基因表达的调控是植物生长发育的关键 所有的有机体。 扰乱正常的控制机制会导致细胞 死亡或转化 在其他情况下，破坏细胞 转录器可以形成病毒感染的基础。 恰当地说，理解真核生物转录的机制是 现代生物学的一个主要目标。 这是一个研究RNA聚合酶II基因转录的建议， 模式生物卡氏阿米巴。 长期目标是 在分子水平上建立真核基因表达的原则。 卡氏阿米巴肌动蛋白基因的体内结构将 被确定。 结果将确定启动子区域， 与蛋白质在体内，并将用作参考点，在体外 问题研究 本研究拟建立一个阿米巴体外转录系统 Castellanii使用阿米巴肌动蛋白I和肌球蛋白II基因。 启动子 肌动蛋白基因体外准确转录所需的元件 将被确定，所需的因素的数量和类型， 转录将被确定。 因子之间的相互作用，RNA 将通过足迹法检查聚合酶II和DNA。 以下步骤 导致引发的preinitiation复杂组件将在 聚合酶和因子结合以及DNA解旋或其他术语 构象变化 转录因子的纯化将是 使用常规和启动子亲和力的组合启动 方法.

The control of gene expression is central to the growth and development of all organisms. Disrupting the normal control mechanisms can lead to cell death or transformation. In other cases, subverting the cells transcription apparatus can form the basis for viral infection. Appropriately, to understand the mechanisms of eukaryotic transcription is a major goal in modern biology. This is a proposal to study transcription of RNA polymerase II genes from the model organism Acanthamoeba castellanii. The long-term goal is to establish principles of eukaryotic gene expression at the molecular level. The in vivo structure of an actin gene from Acanthamoeba castellanii will be determined. The results will identify promoter regions that interact with protein in vivo and will be used as a reference point for in vitro studies. An in vitro transcription system will be developed form Acanthamoeba castellanii using the Acanthamoeba actin I and myosin II genes. Promoter elements required for accurate transcription in vitro from the actin gene will be identified and the number and type of factors required for transcription will be determined. The interaction between factors, RNA polymerase II and DNA will be examined by footprinting. The steps of preinitiation complex assembly that lead to initiation will be defined in terms of polymerase and factor binding as well as DNA unwinding or other conformational changes. Purification of transcription factors will be initiated using a combination of conventional and promoter affinity methods.