Regulation of Transcription:Structure Function Basis

转录调控：结构功能基础

• 批准号: 6620392
• 负责人: AVERELL L GNATT
• 金额: $ 28.62万
• 依托单位: UNIVERSITY OF MARYLAND BALTIMORE
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-02-01 至 2007-01-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6620392
• 关键词: DNA directed RNA polymerase; Escherichia coli; Mammalia; X ray crystallography; chromatography; cryopreservation; crystallization; enzyme model; fungal genetics; fungal proteins; genetic regulation; genetic transcription; intermolecular interaction; model design /development; physical model; protein purification; protein structure function; radiotracer; site directed mutagenesis; species difference; structural biology; transcription factor; yeasts

DESCRIPTION (provided by applicant): The long-term objective of this proposal is to define the mechanisms by which transcription and its regulation are accomplished. These processes underlie basic functions such as morphogenesis and differentiation and are aberrant in cancer cells. As such, the knowledge is fundamental for understanding oncogenesis and oncogenic progression. RNA Polymerase II interacts with many proteins that regulate its function and is the core of the transcription mechanism. During elongation of the mRNA transcript, RNA polymerase II encounters "pause" DNA sequences that appear in at least 40 percent of human open reading frames, causing the enzyme to arrest and preventing completion of mRNA synthesis. The regulatory protein TFIIS binds RNA Polymerase II and plays a crucial role in allowing RNA Polymerase II to complete mRNA synthesis. TFIIS, implicated in tumorigenesis, has a pivotal role in the regulation of transcription. The specific aims of this proposal include; structure determination of yeast RNA Polymerase IITFIIS co-crystals, RNA Polymerase II structure-function relationships and the structure of mammalian RNA Polymerase II. The methodology employed to accomplish these goals involves the purification to homogeneity of yeast and mammalian RNA Polymerase II, as well as yeast TFIIS using ion exchange and affmity chromatography. Upon purification of the proteins, crystals are grown, diffracted in a synchrotron X-ray beam, and the structure determined. Site directed mutagenesis of RNA Polymerase II in yeast will allow for verification and determination of the structure-function relationships of the proteins.

描述（由申请人提供）：该提案的长期目标是定义转录及其法规的机制 成就。这些过程是基本功能（例如形态发生）的基础 和分化，在癌细胞中异常。因此，知识是 理解肿瘤发生和致癌进展的基础。 RNA聚合酶II与许多调节其功能的蛋白质相互作用， 是转录机制的核心。在mRNA的伸长过程中 成绩单，RNA聚合酶II遇到的“暂停” DNA序列出现在 至少40％的人类开放阅读框架，导致酶逮捕 并防止完成mRNA合成。调节蛋白TFIIS结合 RNA聚合酶II并在RNA聚合酶II到达到RNA聚合酶II中起着至关重要的作用 完整的mRNA合成。与肿瘤发生有关的TFII具有关键作用 在转录调节中。 该提案的具体目的包括：酵母的结构测定 RNA聚合酶IITFIIS共结晶，RNA聚合酶II结构功能功能 哺乳动物RNA聚合酶II的关系和结构。方法 实现这些目标的工作涉及净化 酵母和哺乳动物RNA聚合酶II，以及使用离子的酵母TFII 交换和亲和色谱。纯化蛋白质后， 生长晶体，在同步X射线束中衍射，结构 决定。酵母中RNA聚合酶II的定向诱变将允许 用于验证和确定结构功能关系 蛋白质。