INTER AND INTRAMOLECULAR COMMUNICATIONS IN TRANSCRIPTION

转录中的分子间和分子内通讯

• 批准号: 2749959
• 负责人: TOMASZ HEYDUK
• 金额: $ 18.46万
• 依托单位: SAINT LOUIS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1994
• 资助国家: 美国
• 起止时间: 1994-08-01 至 1999-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2749959
• 关键词: DNA directed RNA polymerase; chemical binding; chemical kinetics; computer simulation; conformation; enzyme activity; enzyme complex; enzyme mechanism; enzyme reconstitution; enzyme structure; fluorescence spectrometry; fluorescent dye /probe; genetic promoter element; genetic regulation; genetic transcription; intermolecular interaction; nucleic acid sequence; physical model; protein engineering; protein purification; protein structure function; structural biology; transcription factor

The major unresolved problem in biology is to understand the mechanisms by which transcription is regulated. Such understanding can be accomplished by establishing structural, thermodynamic and kinetic determinants of regulatory processes. This proposal is focused on structural aspects of regulation of transcription by transcription activators. The model system, which will be used in this work, is E coli RNA polymerase (RNAP). In our research we will use a combination of fluorescence spectroscopy, protein chemistry and molecular biology, and we will also take advantage of a novel experimental approach developed recently in our laboratory to map protein domains involved in macromolecular interactions. Our long term goals are to understand how RNAP, depending on the structure of the promoter and depending on the presence or absence of transcription activators readjusts the set of available interactions to perform its biological function. To achieve our goals we propose; aim #1 Studies on the molecular architecture of activator-RNAP-class I and class II promoter complexes to determine contact domains of RNAP involved in protein-protein and protein-DNA interactions, to determine the distances between functional units of the activator-RNAP-promoter complex and their modulation by promoter structure. aim #2 Studies on the role of a specific alpha subunit-DNA contact in RNAP activation by the activator protein to determine whether activator protein-alpha subunit and alpha-subunit-DNA interactions cooperate to produce active form of RNAP. aim #3 Studies on the communication between contact domains of RNAP to determine how DNA binding and promoter melting properties of RNAP are modulated. The results of this study and the novel methodology developed will have general applicability to studies of transcriptional regulation of prokaryotic and eukaryotic systems. Understanding regulation of transcription at the molecular level is of crucial importance for understanding development and the pathogenesis of abnormal development and neoplasia.

生物学中尚未解决的主要问题是通过以下方式来理解机制： 转录是受调控的。 这样的理解是可以实现的 通过建立结构，热力学和动力学的决定因素， 监管程序。 本建议的重点是结构方面， 通过转录激活物调节转录。 模型系统， 将在这项工作中使用的是大肠杆菌RNA聚合酶（RNAP）。 在我们 研究中，我们将使用荧光光谱，蛋白质 化学和分子生物学，我们还将利用一种新的 我们实验室最近开发的一种实验方法， 参与大分子相互作用的结构域。 我们的长期目标是 为了了解RNAP如何依赖于启动子的结构， 取决于转录激活因子的存在与否， 一组可用的相互作用来执行其生物功能。 到 实现我们提出的目标; 目的#1研究I类激活剂RNAP的分子结构， II类启动子复合物，以确定涉及的RNAP的接触结构域 在蛋白质与蛋白质和蛋白质与DNA的相互作用中， 在激活剂-RNAP-启动子复合物的功能单元和它们的 通过启动子结构调节。 目的#2研究特定α亚基-DNA接触在 RNAP通过激活蛋白的激活来确定是否激活 蛋白质-α亚基和α亚基-DNA相互作用协同作用， 产生RNAP的活性形式。 目的#3研究RNAP接触域之间的通信， 确定RNAP的DNA结合和启动子解链特性是如何 调制的 这项研究的结果和开发的新方法将具有 对转录调控研究的一般适用性 原核和真核系统。 了解监管 分子水平的转录对于 了解发育和异常发育的发病机制， 肿瘤形成