Structure/Function of Transcription Complex Regulation

描述(申请人提供)：这个项目的长期目标是确定转录延伸复合体内的相互作用，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。暂停和提前终止不仅会影响细菌基因的表达，正如长期以来所认识到的那样，还会影响大多数人类基因的表达，这些基因是通过将延伸复合体从敏感的启动子近端的暂停或终止切换到能够在通过核小体转录时抵抗终止的有效状态来调节的。在细菌和真核生物中，特殊的调节蛋白都会修改转录复合体，使其抵抗暂停和终止。停顿和终止的基本机制以及调控蛋白控制停顿和终止的机制都依赖于伸长复合体内相互作用的变化，而人们对此知之甚少。这些相互作用的一个中心靶点是将RNA聚合酶中的触发环折叠到触发螺旋中，这是催化核苷酸添加到不断增长的RNA链中所必需的。暂停会干扰触发环路折叠。了解调控蛋白和新生RNA结构如何影响触发环折叠和调控延伸复合体的活性，将提供有关基因调控的关键基础知识，这几乎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，了解它的工作原理有助于识别和表征新的抗生素。生物化学、遗传学和生物物理方法的结合将被用来描述调节调节的伸长复合体中的相互作用。此外，还将研究延长复合体转录核蛋白模板的能力，在核蛋白模板中，类核相关蛋白与细菌细胞中发现的DNA结合。该项目的具体目标是：(I)测试一种被称为桥-螺旋/触发环模型的特定的伸长复合体调控模型，并确定支撑伸长复合体调控的RNA聚合酶的特定变化；(Ii)发现插入一些细菌RNA聚合酶的触发环的一个称为Si3的模块如何参与触发环的功能并调节伸长复合体的活性；(Iii)确定转录终止的机制以及触发环和两种称为NusA和NusG的调节蛋白的具体作用；以及(Iv)确定由一种名为Rho的调节蛋白引起的转录、暂停和终止与类核相关蛋白之间的相互作用。这些研究的影响将是更好地理解延长复合体是如何调节的，在生物技术、人类医学以及原核和真核分子生物学中都有广泛的应用。

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.