Diversity Supplement to Structure/Function of Transcription Complex Regulation to Support Predoctoral Student Christiana Binkley

项目总结/摘要 该项目的长期目标是确定转录延伸复合物内的相互作用， 通过RNA聚合酶引起和控制暂停和终止的调节器。暂停和过早 终止是原核生物和真核生物基因调控许多方面的基础，包括转录 通过染色质和连接到RNA成熟和翻译。暂停的基本机制 和终止以及调节器控制暂停和终止的机制依赖于不良的 理解延伸复合物内相互作用的变化。这些相互作用中的许多调节 RNA聚合酶的构象变化涉及移动的模块，包括钳、触发环和 谱系特异性插入，必须实现有效转录的特定构象。你要明白- 研究调节剂如何促进或抑制这些不同的构象将提供关键的基本知识 指导抗菌药物或基因治疗的调节剂的合理操作。获得的知识 模型细菌系统也有助于理解在转录过程中高度保守的转录机制。 人类此外，细菌RNA聚合酶是抗生素的已知靶点，关于其作用的知识也不多。 功能机制将有助于识别和表征新的抗生素。 结构，生物化学和遗传方法的组合将用于表征相互作用， 延伸复合物中的调节调节作用。低温电子转录检测新方法 显微镜，用于RNA聚合酶与RNA结构，调节剂和 核糖体，并为基因组规模的分析染色质结构和延伸复合物的调控将是 开发这种方法的组合将被用来理解的进展之间的联系， 转录过程中的延伸复合物，细菌染色质的结构，RNA折叠和RNA 翻译.这项工作建立在最近发现的RNA聚合酶协助的结构基础上 RNA折叠，H-NS家族核蛋白丝在刺激暂停和终止中的作用 在转录沉默过程中，以及RNA聚合酶与先驱核糖体的相互作用， 称为expressome。该项目的具体目标是：（一）阐明暂停、终止、 RNA折叠和关键RNAP模块的作用;（ii）定义结构，模式和延伸复合物 H-NS家族核蛋白丝的相互作用;和（iii）确定表达体何时形成以及如何形成 它在转录物延伸期间起作用。这一综合研究将有助于建立一个新的理解跨， 通过定义暂停和终止信号如何改变延伸复合体结构， 活性的动态变化以及染色体结构和翻译偶联如何调节延伸复合体 活动这些研究的影响将是对延伸复合物调控的更好理解， 广泛应用于生物技术、人类医学以及原核和真核分子生物学。

Project Summary/Abstract The long-term goal of this project is to define the interactions within transcription elongation complexes and with regulators that cause and control pausing and termination by RNA polymerase. Pausing and premature termination underlie many aspects of gene regulation in prokaryotes and eukaryotes, including transcription through chromatin and linkages to RNA maturation and translation. Both the basic mechanisms of pausing and termination and the mechanisms by which regulators control pausing and termination depend on poorly understood changes to interactions within the elongation complex. Many of these interactions modulate conformational changes in RNA polymerase involving mobile modules including the clamp, trigger loop, and lineage-specific insertions that must achieve particular conformations for efficient transcription. Understand- ing how regulators promote or inhibit these different conformations will provide key basic knowledge essential to guide the rational manipulation of regulators for antimicrobials or gene therapies. Knowledge gained about model bacterial systems also facilitates understanding of highly conserved mechanisms of transcription in humans. Additionally, bacterial RNA polymerase is a known target of antibiotics, and knowledge about its functional mechanisms will aid in identifying and characterizing new antibiotics. A combination of structural, biochemical, and genetic approaches will be used to characterize the interac- tions in the elongation complex that mediate regulation. New methods for transcription assay by cryo-electron microscopy, for single-molecule assay of RNA polymerase interactions with RNA structures, regulators, and ribosomes, and for genome-scale analysis of chromatin structure and elongation complex regulation will be developed. This combination of approaches will be used to understand connections among progress of the elongation complex during transcription, the structure of bacterial chromatin, RNA folding, and RNA translation. The work builds on recent discoveries of the structural basis by which RNA polymerase assists RNA folding, of the role of H-NS family nucleoprotein filaments in stimulation of pausing and termination during transcriptional silencing, and of interaction of RNA polymerase with the pioneering ribosome in a complex called the expressome. The specific aims of the project are to (i) elucidate steps in pausing, termina- tion, and RNA folding and roles of key RNAP modules; (ii) define structures, patterns, and elongation complex interactions of H-NS family nucleoprotein filaments; and (iii) determine when the expressome forms and how it functions during transcript elongation. This integrated research will help build a new understanding of tran- scriptional regulation by defining how pause and termination signals change elongation complex structure and activity dynamically and how chromosome structure and translational coupling modulate elongation complex activity. The impact of these studies will be an improved understanding of elongation complex regulation, with broad applications to biotechnology, human medicine, and both prokaryotic and eukaryotic molecular biology.