Structure/Function of Transcription Complex Regulation

描述（由申请人提供）：这个项目的长期目标是定义转录延伸复合体内部的相互作用，以及与引起和控制RNA聚合酶暂停和终止的调节蛋白的相互作用。暂停和过早终止不仅影响细菌基因的表达，而且影响大多数人类基因的表达，这些基因是通过将延伸复合物从启动子近端区域的易感性切换到能够抵抗终止的有效状态来调节的。在细菌和真核生物中，专门的调节蛋白修饰转录复合体，使其抵抗暂停和终止。暂停和终止的基本机制以及调节蛋白控制暂停和终止的机制都依赖于延伸复合体内相互作用的变化，而这些变化目前尚不清楚。这些相互作用的中心目标是RNA聚合酶的触发环折叠成触发螺旋，这是催化核苷酸添加到生长的RNA链所必需的。暂停干扰触发回路折叠。了解调控蛋白和新生RNA结构如何影响触发环折叠和调节延伸复合物的活性，将为基因调控提供关键的基础知识，这实际上是人类健康的各个方面的基础。此外，细菌RNA聚合酶是抗生素的已知靶标，了解其工作原理有助于识别和表征新的抗生素。生物化学、遗传和生物物理方法的结合将用于表征介导调节的延伸复合物中的相互作用。此外，延伸复合物转录核蛋白模板的能力，其中核相关蛋白与细菌细胞内发现的DNA结合，将被研究。该项目的具体目标是：(i)测试延伸复合体调节的特定模型，称为桥-螺旋/触发环模型，并定义延伸复合体调节基础上RNA聚合酶的特定变化；（ii）发现插入某些细菌RNA聚合酶的触发环的模块SI3如何参与触发环功能并调节延伸复合物的活性；（iii）确定转录终止的机制以及触发环和两种调节蛋白NusA和NusG的具体作用；（iv）确定由Rho调节蛋白和核相关蛋白引起的转录、暂停和终止之间的相互作用。这些研究的影响将是提高对如何调节延伸复合物的理解，在生物技术，人类医学以及原核和真核分子生物学中具有广泛的应用。

DESCRIPTION (provided by applicant): The long-term goal of this project is to define the interactions within transcription elongation complexes and with regulatory proteins that cause and control pausing and termination by RNA polymerase. Pausing and premature termination affect expression not only of bacterial genes, as long appreciated, but also of most human genes, which are regulated by switching elongation complexes from susceptibility to pausing or termination in the promoter-proximal region to an efficient state able to resist termination while transcribing through nucleosomes. In both bacteria and eukaryotes, specialized regulatory proteins modify the transcription complex to make it resistant to pausing and termination. Both the basic mechanisms of pausing and termination and the mechanisms by which regulatory proteins control pausing and termination depend on changes to interactions within the elongation complex that are poorly understood. A central target of these interactions is folding of the trigger loop in RNA polymerase into the trigger helices, which is required to catalyze addition of nucleotides to the growing RNA chain. Pausing interferes with trigger loop folding. Understanding how regulatory proteins and nascent RNA structures influence trigger-loop folding and regulate the activity of elongation complexes will provide key basic knowledge about gene regulation, which underlies virtually every aspect of human health. Further, bacterial RNA polymerase is a known target of antibiotics, and knowledge about how it works aids in identifying and characterizing new antibiotics. A combination of biochemical, genetic, and biophysical approaches will be used to characterize the interactions in the elongation complex that mediate regulation. Additionally, the ability of elongation complexes to transcribe nucleoprotein templates in which nucleoid-associated proteins are bound to DNA as found within bacterial cells will be studied. The specific aims of the project are to (i) test a specific model of elongation complex regulation known as the bridge-helix/trigger-loop model and to define specific changes in RNA polymerase that underlie elongation complex regulation; (ii) discover how a module that inserts in the trigger loop of some bacterial RNA polymerases, called SI3, participates in trigger-loop function and modulates the activity of elongation complexes; (iii) determine the mechanism of transcription termination and the specific roles of the trigger loop and two regulatory proteins called NusA and NusG; and (iv) determine the interplay among transcription, pausing, and termination caused by a regulatory protein called Rho, and nucleoid-associated proteins. The impact of these studies will be an improved understanding of how elongation complexes are regulated, with broad applications to biotechnology, human medicine, and both prokaryotic and eukaryotic molecular biology.