INTER AND INTRAMOLECULAR COMMUNICATIONS IN TRANSCRIPTION

生物学中尚未解决的主要问题是通过 哪种转录是受调控的。这样的理解是可以实现的 通过建立结构、热力学和动力学决定因素 监管流程。本提案的重点是结构方面的 转录激活剂对转录的调节。模型系统， 这项工作中将使用的是大肠杆菌RNA聚合酶(RNAP)。在我们的 研究中我们将结合使用荧光光谱、蛋白质 化学和分子生物学，我们还将利用一种新的 我们实验室最近发展起来的蛋白质图谱的实验方法 参与大分子相互作用的结构域。我们的长期目标是 要了解RNAP是如何根据启动子和 根据转录激活子的存在或不存在而重新调整 执行其生物功能的一组可用相互作用。至 实现我们提出的目标； 目的1研究激活剂-RNAP-I类和 确定RNAP涉及的接触结构域的II类启动子复合体 在蛋白质-蛋白质和蛋白质-DNA相互作用中，确定 激活剂-RNAP-启动子复合体的功能单元与其 启动子结构的调控。 目的#2研究特定的α亚基-DNA接触在 RNAP激活由激活剂蛋白决定是否激活剂 蛋白质-α亚基和α-亚基-DNA相互作用共同作用 产生活性形式的RNAP。 目的#3研究RNAP接触域间的通信 确定RNAP的DNA结合和启动子熔融特性 调制过的。 这项研究的结果和开发的新方法将具有 转录调控研究的一般适用性 原核生物和真核生物系统。了解监管规则 在分子水平上的转录对于 对发育异常的认识及其发病机制 肿瘤。

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.