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

转录复合体调节结构/功能的多样性补充以支持博士生克里斯蒂娜·宾克利

• 批准号: 10351034
• 负责人: Robert Landick
• 金额: $ 1.46万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-07-01 至 2023-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10351034
• 关键词: Abbreviations; Active Sites; Address; Amino Acids; Antibiotics; Antisense RNA; Bacterial Chromosomes; Bacterial Model; Bacterial RNA; Binding; Biochemical; Biochemical Genetics; Biochemistry; Biogenesis; Biological Assay; Biotechnology; C-terminal; Cells; Chromatin; Chromatin Structure; Chromosome Structures; Closure by clamp; Complex; Coupled; Coupling; Cryoelectron Microscopy; Cytidine; DNA; DNA Insertion Elements; DNA-Directed RNA Polymerase; Diffuse; Disease; Escherichia coli; Eukaryota; Family; Filament; Fluorescence; Funding; Gene Expression Regulation; Gene Silencing; Genetic Transcription; Goals; Health; Human; In Vitro; Knowledge; Learning; Maps; Mediating; Medicine; Methods; Modeling; Molecular; Molecular Biology; Molecular Chaperones; Molecular Conformation; Mycobacterium tuberculosis; N-terminal; Nucleoproteins; Nucleotides; Orthologous Gene; Pattern; Phenylalanine; Prokaryotic Cells; Protein Biosynthesis; RNA; RNA Folding; RNA chemical synthesis; Regulation; Reporter; Research; Resolution; Ribosomes; Role; Saccharomyces cerevisiae; Signal Transduction; Stress; Structure; Students; System; Thermus thermophilus; Time; Transcript; Transcription Elongation; Transcriptional Regulation; Translations; Triplet Multiple Birth; Work; antimicrobial; base; ds-DNA; gene therapy; genetic approach; genome-wide; improved; in vivo; insight; microbial community; novel; pathogenic microbe; pre-doctoral; premature; single molecule; structural biology; time use; tool; virtual; Abbreviations; Active Sites; Address; Amino Acids; Antibiotics; Antisense RNA; Bacterial Chromosomes; Bacterial Model; Bacterial RNA; Binding; Biochemical; Biochemical Genetics; Biochemistry; Biogenesis; Biological Assay; Biotechnology; C-terminal; Cells; Chromatin; Chromatin Structure; Chromosome Structures; Closure by clamp; Complex; Coupled; Coupling; Cryoelectron Microscopy; Cytidine; DNA; DNA Insertion Elements; DNA-Directed RNA Polymerase; Diffuse; Disease; Escherichia coli; Eukaryota; Family; Filament; Fluorescence; Funding; Gene Expression Regulation; Gene Silencing; Genetic Transcription; Goals; Health; Human; In Vitro; Knowledge; Learning; Maps; Mediating; Medicine; Methods; Modeling; Molecular; Molecular Biology; Molecular Chaperones; Molecular Conformation; Mycobacterium tuberculosis; N-terminal; Nucleoproteins; Nucleotides; Orthologous Gene; Pattern; Phenylalanine; Prokaryotic Cells; Protein Biosynthesis; RNA; RNA Folding; RNA chemical synthesis; Regulation; Reporter; Research; Resolution; Ribosomes; Role; Saccharomyces cerevisiae; Signal Transduction; Stress; Structure; Students; System; Thermus thermophilus; Time; Transcript; Transcription Elongation; Transcriptional Regulation; Translations; Triplet Multiple Birth; Work; antimicrobial; base; ds-DNA; gene therapy; genetic approach; genome-wide; improved; in vivo; insight; microbial community; novel; pathogenic microbe; pre-doctoral; premature; single molecule; structural biology; time use; tool; virtual

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.

项目摘要/摘要 该项目的长期目标是定义转录伸长络合物中的相互作用和 与导致和控制RNA聚合酶暂停和终止的调节剂。暂停和过早 终止是基因调节的许多方面的原核生物和真核生物，包括转录 通过染色质和与RNA成熟和翻译的联系。暂停的两种基本机制 和终止以及监管机构控制暂停和终止的机制取决于较差 理解对延伸复合物中相互作用的变化。这些相互作用中的许多调节 RNA聚合酶的构象变化，涉及移动模块，包括夹具，触发环和 谱系特异性插入必须达到有效转录的特定构象。理解- 监管机构如何促进或抑制这些不同的构象将提供关键的基本知识必不可少的 指导对抗菌药物或基因疗法的调节剂的合理操纵。知识获得了 模型细菌系统还促进了对高度保守的转录机制的理解 人类。此外，细菌RNA聚合酶是抗生素的已知靶标，及其知识 功能机制将有助于识别和表征新的抗生素。 结构，生化和遗传方法的结合将用于表征 介导调节的延伸络合物中的tions。通过冷冻电子转录测定的新方法 显微镜，用于与RNA聚合酶相互作用与RNA结构，调节剂和 核糖体，以及用于染色质结构和伸长复合物调控的基因组规模分析 发达。这种方法的组合将用于了解 转录过程中的伸长复合物，细菌染色质，RNA折叠和RNA的结构 翻译。这项工作建立在最新的RNA聚合酶有助于的结构基础的基础上 RNA折叠，H-NS家族核蛋白丝在暂停和终止中的作用 在转录沉默期间，以及RNA聚合酶与A中的核糖体的相互作用 复杂称为Expressome。该项目的具体目的是（i）阐明暂停的步骤 关键RNAP模块的tion和RNA折叠和作用； （ii）定义结构，模式和伸长率复合物 H-NS家族核蛋白丝的相互作用； （iii）确定何时表格以及如何 它在笔录伸长过程中起作用。这项综合研究将有助于建立对Tran的新理解 通过定义暂停和终止信号如何改变伸长复杂结构和 动态活性以及染色体的结构和翻译耦合如何调节伸长复合物 活动。这些研究的影响将是对伸长复合复杂调节的一种改进，并与 在生物技术，人类医学以及原核生物和真核分子生物学上的广泛应用。